Method for separating an analyte from a sample

ABSTRACT

An analyte is separated from a fluid sample by introducing the sample into a cartridge having a sample port and a first flow path extending from the sample port. The first flow path includes an extraction chamber containing a solid support for capturing the analyte from the sample. The cartridge has a second flow path for eluting the captured analyte from the extraction chamber, the second flow diverging from the first flow path after passing through the extraction chamber. The sample is forced to flow through the extraction chamber and into a waste chamber, thereby capturing the analyte with the solid support as the sample flows through the extraction chamber. The captured analyte is then eluted from the extraction chamber by forcing an elution fluid to flow through the extraction chamber and along the second flow path.

PARENT APPLICATION DATA

This application is a divisional of U.S. patent application Ser. No.11/121,541, filed May 3, 2005, now U.S. Pat. No. 7,569,346, which is adivisional of U.S. patent application Ser. No. 10/005,685, filed Nov. 7,2001, now, U.S. Pat. No. 6,893,879, issued on May 17, 2005, which is adivisional of U.S. patent application Ser. No. 09/331,911 filed Jun. 25,1999, now U.S. Pat. No. 6,440,725 issued on Aug. 27, 2002, which is anational stage entry (371) of International Application PCT/US98/27632filed Dec. 24, 1998 which international application claims priority fromU.S. patent application Ser. No. 09/115,454 filed Jul. 14, 1998, nowabandoned, and from U.S. patent application Ser. No. 08/998,188 filedDec. 24, 1997, now abandoned. All of these applications are incorporatedby reference herein for all purposes.

FIELD OF THE INVENTION

This invention relates to a method for separating an analyte from asample.

BACKGROUND OF THE INVENTION

The analysis of clinical or environmental fluids generally involves aseries of chemical, optical, electrical, mechanical, or thermalprocessing steps on the fluid samples. Whether incorporated into abench-top instrument, a disposable cartridge, or a combination of thetwo, such processing involves complex fluidic assemblies and processingalgorithms.

Contemporary biomedical processing instruments are typically complex,robotically operated devices that move boluses of liquids automaticallyfrom one processing region to another. Prior cartridges have alsogenerally processed a fluid sample as a fluid plug or bolus, moving asmall quantity of sample from one region to another, where a furtherprocess is conducted. For example, Anderson et al. disclose such adevice for sample processing in an article entitled “MicrofluidicBiochemical Analysis System”, Transducers '97, 1997 InternationalConference on Solid-State Sensors and Actuators, Chicago, Jun. 16-19,1997, pg. 477-480.

In many analytical procedures, relatively large volumes of liquid (frommicroliters to milliliters) must be analyzed. Using the bolus approach,such volumes must be held in a container while each operation isperformed. While the bolus approach allows for the implementation ofcomplex processing methods, the volume of the fluid sample which can beprocessed is limited by the size of the individual processing regions,especially where the sample is transiently processed. Thus, the lowestdetectable concentration of analyte, i.e. sensitivity, in any assaybased on a bolus approach is also limited.

If the container is fabricated with integrated circuit chip technologies(microfluidic chips), the microfabricated chip must be very large toaccommodate the relatively large volumes needed to detect a lowconcentration of analyte. For example, for a 100 microliter volume, achip at least 1 cm on a side would be required for each bolus processingregion. Such a large chip would not only be expensive, but would alsodefeat the purpose of miniaturization, especially for many types ofdisposable medical or environmental diagnostic uses.

Present day microfluidic technology has focused on picoliter, nanoliter,and microliter fluid volumes. These small volumes are not practical formany realistic diagnostic applications. As shown in FIG. 1, the fullrange of chemical concentrations which one may want to detect inbiological samples spans at least 20 orders of magnitude (from 6copies/mL to 6×10²⁰ copies/mL). Therefore, a cartridge for detecting thefull range of potential analytes (especially DNA which exists in verylow concentration in most biological samples) should be capable ofprocessing large as well as small sample volumes.

Of special interest is the detection of low copy concentrations ofanalytes such as DNA, in which case large sample volumes are required.For example, the minimum theoretically detectable concentration for DNAprobe assays necessitates large sample sizes, such as about 10⁻⁴ litersor more. In detecting infectious diseases, gram negative bacteria can bepresent at less than 10 copies per milliliter of blood, cryptosporidiumgenerally appears as only a few copies per gallon of drinking water,concentrated biothreat agents, e.g. anthrax, at less than 100 copies permilliliter of water, and food poisoning agents, such as E. coli andsalmonella, may be manifested in less than 10 copies per gram of food.

Thus, sample volumes needed to detect such infectious disease analyteswould be larger than those required for detecting analytes present inhigher concentrations, as in most clinical and immunochemistry assays.In addition, in the case of more concentrated analytes, such as those inimmunoassays and clinical chemistry assays, a large volume sampleprovides more options for choosing less sensitive detection means, aswell as the ability to divide the sample and detect multiple analytes.On the other hand, despite the merits of large sample volumes, it isgenerally recognized that unique functions can be realized withmicrofluidic structures, which are generally not compatible with largevolumes.

SUMMARY

The processing devices and methodology of the present inventionelegantly resolve the dilemma between large sample volumes andmicrofluidic structures by incorporating microfluidic chips orcomponents into larger cartridges having any desired combination ofmicroscale to macroscale channels, chambers, reservoirs, detection andprocessing regions. This makes it possible to exploit the key attributesof microfabricated chips and other miniature fluidic or analyticalcomponents in a conventional, cartridge-type, physical environment. Sucha combination, while superficially less sophisticated than“lab-on-a-chip” technology, affords a superior blend of efficiency andconvenience in design, manufacture, and use.

In a preferred embodiment, the invention provides a device forseparating a desired analyte from a fluid sample and for concentratingthe analyte into a volume of elution fluid smaller than the originalsample volume. The desired analyte may comprise, e.g., organisms, cells,proteins, nucleic acid, carbohydrates, virus particles, bacterias,chemicals, or biochemicals. In a preferred use, the desired analytecomprises nucleic acid.

The device comprises a cartridge having formed therein an inlet port forintroducing the sample into the cartridge and a sample flow pathextending from the inlet port through the body of the cartridge. Thesample flow path includes an analyte capture region having at least oneflow-through component for capturing the desired analyte from thesample.

The flow-through component is preferably a microfabricated chip having achamber with internal microstructures formed therein. Themicrostructures have sufficiently high surface area and binding affinitywith the desired analyte to capture the analyte as the sample flowsthrough the chip. The microstructures preferably comprise an array ofcolumns integrally formed with at least one wall of the chamber andextending into the chamber. In an alternative embodiment, theflow-through component comprises a channel or chamber in the cartridgecontaining at least one solid support for capturing the analyte.Suitable solid supports include, e.g., filters, beads, fibers,membranes, glass wool, filter paper, polymers and gels.

A flow path for carrying elution fluid is also formed in the cartridge.The elution flow path passes through the flow-through component, therebyreleasing captured analyte from the component into the elution fluid.The elution flow path diverges from the sample flow path after passingthrough the component. In the preferred embodiment, the cartridge alsoincludes, or may be coupled to, a heating element for heating thecomponent, thereby increasing elution efficiency.

The cartridge also includes at least one flow controller, e.g., one ormore valves, flow diverters, or fluid diodes, for directing the fluidsample into the sample flow path after the sample flows through thecapture component and for directing the elution fluid and eluted analyteinto the elution flow path after the elution fluid flows through thecapture component. In the preferred embodiment, the cartridge furtherincludes a waste chamber at the end of the sample flow path forcollecting the remaining fluid sample and a second chamber at the end ofthe elution flow path for receiving the eluted analyte. The secondchamber may alternatively be a reaction chamber formed in a separatereaction vessel coupled to the cartridge to receive the eluted analytefor further processing.

In contrast to prior fluidic cartridges that process a fluid sample as abolus, the continuous-flow cartridge of the present invention permitsthe rapid processing of a fluid sample that is larger in volume than anyinteractive region within the cartridge. The ability to process largersample volumes allows increased sensitivity in the detection of low copyconcentrations of analytes, such as nucleic acid.

In a preferred mode of operation, the cartridge is used to separatenucleic acid, e.g. DNA or RNA, from a fluid sample and to concentratethe nucleic acid into a smaller volume of elution fluid. In theseapplications, it is preferred that the sample flow path formed in thecartridge include a lysing region, e.g. a channel or chamber, for lysingcells, spores, or microorganisms in the fluid sample. Preferably, anultrasonic transducer, such as an ultrasonic horn, is coupled to thecartridge for transferring ultrasonic energy to the fluid sample in thelysing region, thereby effecting lysis of the cells, spores, ormicroorganisms. The lysing channel or chamber may additionally includeparticles or beads for rupturing the cells, spores, or microorganisms asthe ultrasonic energy is applied.

The lysing channel or chamber preferably contains a solid phase forcapturing the cells, spores, or microorganisms as the sample flowsthrough the chamber. Suitable solid phases include, e.g., filters,beads, fibers, membranes, glass wool, filter paper, polymers and gels.Lysing is accomplished by applying ultrasonic energy to the cells,spores, or microorganisms captured on the solid phase. The ultrasonicenergy may be supplied from, e.g., an ultrasonic horn coupled to a wallof the lysing chamber or built into the cartridge. The cartridge mayalso contain, or be coupled to, a heating element in thermal contactwith the lysing chamber for heating the fluid sample as the ultrasonicenergy is applied.

In another embodiment of the cartridge, the lysing region comprises alysing chamber positioned upstream of the capture region, and thecartridge further includes a reagent chamber in fluid communication withthe lysing chamber for holding a lysing reagent. In this embodiment, afluid motive source, such as a pump, is also provided for forcing thelysing reagent to flow into the lysing chamber to contact the sample.Lysing reagents may also be used in combination with the ultrasoniclysing embodiments described above.

In the preferred embodiment, the invention also provides an externalinstrument for receiving one or more of the cartridges. The externalinstrument includes a fluid motive source, e.g., one or more pumps,vacuums, or pressure sources, that interface with one or more ports orvents formed in the cartridge, to force the sample to flow through thecartridge. Either the instrument or the cartridge may also includeprocessing electronics, e.g., one or more microprocessors,microcontrollers, or memory chips, for controlling the operation of thecartridge.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot of analyte concentration (copy number) versus samplevolume showing the minimum volume required for statistically significantdetection of analyte.

FIG. 2 is a schematic, plan view of a cartridge for processing a fluidsample according to a first embodiment of the invention.

FIG. 3 is a perspective view of an instrument holding several cartridgesfor processing.

FIG. 4 is an exploded view of a fluid diode for the prevention ofbackflow.

FIG. 5A is a schematic, plan view of an electrolytic pump.

FIG. 5B is a schematic side view of the pump of FIG. 5A.

FIG. 6 is a schematic, cross sectional view of a flow-through chip forextracting analyte from a fluid sample according to a preferredembodiment of the invention.

FIG. 7 is a bottom plan view of the chip of FIG. 6.

FIG. 8 is a three-dimensional view of microcolumns formed in anextraction chamber of the chip of FIG. 6.

FIG. 9 is a schematic, plan view of the microcolumns in the chip of FIG.6.

FIG. 10 is a plan view of two adjacent microcolumns in the chip of FIG.6.

FIG. 11 is a schematic view of an etch mask defining a chamber patternand a column pattern used in the fabrication of the chip of FIG. 6.

FIG. 12 is a schematic, cross sectional view of an alternativemicrofabricated chip for extracting analyte from a fluid sample.

FIG. 13 is a schematic, cross sectional view of another microfabricatedchip for extracting analyte from a fluid sample.

FIG. 14 is a schematic, cross sectional view of a microfabricated chipfor extracting analyte from a fluid sample according to a furtherembodiment of the invention.

FIG. 15 is a partially exploded, cross-sectional view of amicrofabricated chip embedded in a plastic cartridge.

FIG. 16 is a partially exploded view of another cartridge showing abottom plate, interactive regions, connecting channels, flex circuitry,fluid pouches, and a top plate with a fluid inlet port.

FIG. 17 is a cross-sectional view of a region of the cartridge of FIG.16 containing filter paper for capturing analyte.

FIG. 18 is a schematic view of a flow diverter region of the cartridgeof FIG. 16.

FIG. 19 is a schematic, side view of an ultrasonic horn coupled to acartridge for lysing of sample components according to anotherembodiment of the invention.

FIG. 20 is a schematic side view of an ultrasonic transducer coupled toa cartridge containing beads for lysing of sample components accordingto a further embodiment of the invention.

DETAILED DESCRIPTION

The present invention provides a cartridge for performing variousoperations on a fluid sample as the sample flows through a series ofinterconnected, interactive regions within the cartridge. The regionsare located sequentially along a fluid flow path through the cartridge,so that a segment of the fluid stream is exposed to a specific operationat one region, then another operation at the next region, etc. Thesample flows through the interactive regions so that it issimultaneously in contact with more than one region at a given time. Thesample flow is preferably continuous, so that the operations at eachregion occur simultaneously and sequentially on the fluid stream.

The cartridges of the present invention allow for significantly improvedprocessing of a fluid sample for the detection and/or analysis ofchemical components in the sample, such as biological molecules. Apioneering improvement over the prior art is the ability to rapidlyprocess a fluid sample that is larger in volume than any interactiveregion within the cartridge, thereby permitting increased sensitivity inthe detection of low copy concentrations of analytes, such as nucleicacid. The cartridges may also be designed to automatically conductprocesses, such as mixing reagents with the fluid sample, lysing,filtering, and introducing the mixture into a reaction chamber orseparate reaction vessel appropriate for further processing, e.g.,detection or amplification of the analyte.

Since the operations on the fluid sample are performed on the samplestream as it flows through the various regions of the cartridge, anyincorporated microfluidic processing chip or other component can be verysmall, as much as one hundred times smaller than with the bolus-orientedapproach. This allows the entire processing facility to be small, yetcapable of processing relatively large fluid samples (e.g., 0.1 to 10mL), and thus to take advantage of the unique properties of very smallmicrofluidic chips or other fluid processing components.

In a preferred embodiment, the invention provides a device forseparating a desired analyte from a fluid sample and for concentratingthe analyte into a volume of elution fluid smaller than the originalsample volume. The desired analyte may comprise, e.g., organisms, cells,proteins, nucleic acid, carbohydrates, virus particles, bacterias,chemicals, or biochemicals. In a preferred use, the desired analytecomprises nucleic acid.

As used herein, the term “nucleic acid” refers to any synthetic ornaturally occurring nucleic acid, such as DNA or RNA, in any possibleconfiguration, i.e., in the form of double-stranded nucleic acid,single-stranded nucleic acid, or any combination thereof. As usedherein, the term “fluid sample” includes both gases and liquids,preferably the latter. The fluid sample may be an aqueous solutioncontaining particles, cells, microorganisms, ions, or small and largemolecules, such as proteins and nucleic acids, etc. In a particular use,the fluid sample may be a bodily fluid, e.g., blood or urine, or asuspension, such as pulverized food. The fluid sample may be pretreated,for example, mixed with chemicals, centrifuged, pelleted, etc., or thefluid sample may be in a raw form.

FIG. 2 shows an example of a cartridge 101 according to a preferredembodiment of the invention. The cartridge is designed to process afluid sample and amplify nucleic acids, such as by polymerase chainreaction (PCR). The cartridge 101 includes a sample port 103 forintroducing a fluid sample into the cartridge and a sample flow pathextending from the port 103 into the body of the cartridge.

The sample flow path includes a channel 105 leading from the sample port103 to a mixing chamber 107 for mixing of the sample with lysingreagents. The sample flow path also includes a lysing chamber 119 wherethe sample contacts a filter to capture components, e.g., cells, spores,or microorganisms in the sample. The captured components are lysed inchamber 119. The sample flow path further includes a flow-throughcomponent 122 for capturing a desired analyte, e.g. nucleic, acid, fromthe sample as the sample flows through the component 122.

The flow-through component 122 is preferably a microfabricated chiphaving a chamber with internal microstructures formed therein. Themicrostructures have sufficiently high surface area and binding affinitywith the desired analyte to capture the analyte as the sample flowsthrough the chip. The microstructures preferably comprise an array ofcolumns integrally formed with at least one wall of the chamber andextending into the chamber. Various embodiments of the microfabricatedchip are described in detail below with reference to FIGS. 6-14.

In an alternative embodiment, the flow-through component 122 comprises achannel or chamber formed in the cartridge. The channel or chambercontains at least one solid support for capturing the desired analytefrom the fluid sample as the sample flows through the solid support.Suitable solid supports include filters, beads, fibers, membranes, glasswool, filter paper, polymers and gels.

The sample flow path also includes a channel 135 leading to flowcontrollers 41A and 41B, and a channel 136 leading to a vented wastechamber 139. The flow controllers 41A and 41B are arranged to direct thesample into the waste chamber 139 after the sample flows through thecapture component 122. The flow controllers 41A and 41B may be, e.g.,valves, flow diverters, or fluid diodes.

A flow path for carrying elution fluid is also formed in the cartridge101. In the preferred embodiment, the cartridge includes a storagechamber 127 for storing elution fluid. The elution flow path extendsfrom the chamber 127 through a channel 131 and passes through theflow-through component 122, thereby releasing captured analyte from thecomponent into the elution fluid. In an alternative embodiment, thecartridge includes a separate inlet port, in place of or in addition tothe storage chamber 127, for introducing elution fluid into thecartridge from an external source.

The elution flow path diverges from the sample flow path after passingthrough the component 122. In this example, the elution flow pathfollows the channel 135 to the flow controllers 41A and 41B. The flowcontrollers 41A and 41B are arranged to direct the elution fluid andeluted analyte into a reagent chamber 141 containing PCR reagents. Thereagent chamber 141 is in fluid communication with a reaction chamber143 for PCR amplification.

The reaction chamber 143 may be a chamber formed in the cartridge 101.Alternatively, the reaction chamber 143 may be formed in a separatereaction vessel designed to be coupled to the cartridge to receive theeluted analyte. Suitable reaction vessels for this purpose are disclosedin International Application Number PCT/US98/03962 filed Mar. 2, 1998and entitled “Heat Exchanging, Optically Interrogated Chemical ReactionAssembly”, the disclosure of which is incorporated by reference herein.The application also teaches a thermal sleeve for receiving andthermally cycling the reaction chamber. For this reason, it isadvantageous for the reaction chamber to protrude from the rest of thecartridge body to facilitate insertion of the reaction chamber into thethermal sleeve.

The cartridge 101 also includes a storage chamber 109 for storing alysing reagent, and a storage chamber 125 for storing a washing reagent.The cartridge 101 further includes flow controllers 123, such as valvesor fluid diodes, for controlling the flow of fluid through thecartridge. The cartridge 101 also preferably includes resistive sensors115 for sensing the presence of fluid in various channels and regions.

Referring to FIG. 3, the cartridge 101 is preferably used in combinationwith a portable, i.e. hand-held or desk-top, external instrument 211designed to accept one or more of the cartridges 101. The connectionbetween the disposable cartridge 101 and the external instrument 211 ispreferably by means of a thin, card-like section of the cartridge 101,and a mating connector within the instrument 211. This type ofconnection is similar to the standard card edge connectors used withprinted circuit boards in, e.g., personal computers or card cages.

As shown in FIG. 2, narrow fingers 151 of conductive material on thecard or on foil come in contact with gold connectors in the instrumentas the cartridge 101 is inserted for processing. Many connections can bemade within a small width of cartridge in this implementation. In thecase of the cartridge, the card may be a thin section of molded plasticor a sheet on which conductive materials are deposited.

Electrical connections may also be used to transfer information to andfrom stored memory and/or intelligence on the cartridge 101. Forexample, a memory or microprocessor chip may be incorporated as part ofthe cartridge. This chip preferably contains information such as thetype of cartridge, program information such as specific protocols forthe processing of the cartridge, tolerances for accept and reject,serial numbers and lot codes for quality tracking, and provision forstoring the results of the processing.

Integrated electronic memory on the cartridge 101 allows for rapid,easy, and error-free set-up of the instrument 211 for different fluidicprocessing protocols. When a cartridge is inserted into the instrument,the instrument may electronically address the memory on the cartridge,and thus automatically receive the appropriate set of instructions forcontrolling the time-sequence of fluidic operations to be carried outwith the inserted cartridge. The instrument 211 may simply sequentiallyretrieve and execute each step in the cartridge's memory, or downloadits contents so that the user may edit the sequence using, e.g.,keyboard 213.

If suitable memory is included on the cartridge, such as writable memory(e.g., erasable programmable read-only memory (EPROM), electricallyerasable programmable read-only memory (EEPROM), etc., intermediate andfinal results, based on the sample introduced into the cartridge, couldbe written by the instrument into the cartridge's memory for co-locatedstorage with the physical sample after processing. This is particularlyadvantageous in applications where archiving of samples and results isnecessary, such as forensics.

In addition, other information can be stored in the memory on thecartridge, in unalterable (or alterable) forms. For example, cartridgeserial number, lot manufacture information, and related informationcould be pre-programmed and unalterable. User data, technicianidentification number, date of test, location of test and instrumentserial number could be unalterably written into the cartridge. Thisallows for easy identification of the “chain of custody” in the handlingof a specimen. Engineers skilled in the art of data storage willrecognize that other memory means than electronic can be used, such asoptically-addressed printed regions (e.g., ink-jet or thermal), magneticstrips, etc.

Electrical power may be provided to the cartridge 101 from the externalinstrument 211. Alternatively, instead of making the instrument bulkierand heavier by adding batteries to accommodate the power needs ofmultiple cartridges used sequentially to process many samples, the powersource for each cartridge may be included on the cartridge, sufficientto power the instrument and cartridge.

The instrument 211 preferably includes processing electronics, e.g., oneor more microprocessors, multiplexers, power control circuits, andsensor circuits, for controlling the operation of the cartridge 101. Theprocessing electronics are connected by the contact fingers 151 andelectrical leads 147 to various regions, storage areas, pumps, sensors,and channels in the cartridge 101. Alternatively, there may be otherdata links of the cartridge to the instrument, such as radio frequencyor infrared links. Although the processing electronics are physicallylocated in the external instrument 211 in the preferred embodiment, itis to be understood that the processing electronic may also be locatedon the cartridge 101.

Both external and internal fluid motive sources are suitable for usewith the cartridges disclosed herein. The fluid motive source may becontained in or on the cartridge 101 itself, or may be external to thecartridge, e.g., included in the external instrument 211 into which thecartridge 101 is inserted for processing. One type of fluid motivesource described in this disclosure is an electrolytic pump (e-pump)located inside the cartridge 101. The fluid inside a sealed pouch isdecomposed into gaseous elements by an electrical current, therebypressurizing and expanding the pouch. This sealed pumping pouch, ore-pump, is positioned against a reagent pouch and forces the contents ofthe reagent pouch into the fluidic circuit as the pumping pouch expands.

Other types of fluid motive sources may also be used with the cartridgesof the present invention. For example, a stepper motor or solenoid canbe used to provide a force and press against a reagent pouch inside thecartridge, thereby forcing the contents of the reagent pouch into thefluidic circuit. Alternatively, a mechanical spring located eitherinside the cartridge or inside the external instrument may provide themotive source for pressing on the reagent pouch and forcing the reagentinto the fluidic circuit. The mechanical energy stored in the spring mayeither be built into the cartridge during manufacture or be generatedduring insertion of the cartridge into the instrument (i.e. cocking thespring during manual insertion of the cartridge).

Other potential fluid motive sources include a pneumatic pressure source(or vacuum source) located inside the cartridge or inside theinstrument. Such a fluid motive source may be provided by a pressurized(or evacuated) canister, chip, or other container. The motive sourcecould also be a compressor or vacuum pump located either inside thecartridge or inside the instrument. In the instances in which anexternal pressure or vacuum motive source is used, the cartridge hassuitable ports, vents, or channels for interfacing with the source.Likewise, electrophoretic or electroosmotic sources may be employed.Piezoelectrically, magnetically, or electrostatically driven membranepumps or valves could also be incorporated into the cartridge orpermanently installed in the instrument so that the devices aremechanically interfaced with the cartridge when the cartridge isinserted into the instrument.

In operation, a fluid sample containing a desired analyte. e.g. nucleicacid, is added to the sample port 103 of the cartridge 101 and forced toflow continuously (such as with an electrolytic or mechanical pump) downa channel 105 and into the mixing chamber 107. Lysing reagents aresimultaneously released from the storage chamber 109 and forced to flowdown a channel 111 and into the chamber 107. Suitable lysing reagentsinclude, for example, solutions containing a chaotropic salt, such asguanidine HCl, guanidine thiocyanate, guanidine isothiocyanate, sodiumiodide, urea, sodium perchlorate, and potassium bromide.

The fluid sample and lysing reagents traveling in the channels 105 and111, respectively, are detected by resistive sensors 115. As the lysingreagent contacts the fluid sample, cells, spores, or microorganismspresent in the fluid sample begin to be lysed. The fluid sample andlysing reagent continue to flow into the lysing chamber 119 where thesample contacts a filter and the cells, spores, or microorganisms arecaptured. The lysing reagent continues to lyse the captured samplecomponents. The filter also serves to remove debris from the fluidsample. In another important embodiment of the invention, an ultrasonictransducer is coupled to the cartridge 101 next to lysing chamber 119,e.g. coupled to a wall of the chamber 119, and the sample components arelysed by ultrasonic energy provided by the transducer. Variousultrasonic lysing embodiments are discussed in greater detail below withreference to FIGS. 19-20.

The lysed sample proceeds from the lysing chamber 119 down channel 121and is forced to flow through the capture component 122. As the fluidsample and lysing reagent flow through the component 122, nucleic acidin the fluid sample binds to the component 122. The flow-rate of thefluid sample through the component 122 is preferably in the range of 0.1to 50 μL/sec. The fluid sample and lysing reagent exiting the component122 flow down channel 135, through the flow controller 41A, and throughchannel 136 to the waste chamber 139. In another embodiment, afterflowing through the component 122, the fluid sample may be redirected torecirculate through the component additional times.

After the fluid sample is forced to flow through the component 122, thewashing reagent in storage region 125 is forced to flow down a channel129 and through the component 122. The wash flow rate is preferably onthe range of 0.5 to 50 μL/sec. Fluid is prevented from flowing upstreamin the cartridge by flow controllers 123 in channels 121, 129, and 131.The washing reagent washes residual contaminants, such as chaotropicsalts, from the component 122. A variety of suitable wash solutions ofvarying pH, solvent composition, and ionic strength may be used for thispurpose and are well known in the art. For example, a suitable washingreagent is a solution of 80 mM potassium acetate, 8.3 mM Tris-HCl, pH7.5, 40 uM EDTA, and 55% ethanol.

The washing reagent continues to flow through the flow controller 41Aand into the waste chamber 139.

After washing the component 122, elution fluid from the storage region127 is forced to flow down channel 131 and through the component 122,thus releasing the nucleic acid from the component into the elutionfluid. At this point, the flow controllers 41A and 41B are reconfiguredto prevent the elution fluid from flowing through the flow controller41A and to permit the elution fluid to flow through the flow controller41B into the reagent chamber 141. The flow rate of elution fluid throughthe component 122 is preferably in the range of 0.1 to 10 μL/sec. Theflow rate of the elution fluid may be relatively slow as compared to theflow rate of the fluid sample to allow for more analyte to be releasedfrom the component.

In general, any suitable elution fluid may be used to elute nucleic acidfrom the component 122. Such elution fluids are well known in the art.For example, the elution fluid may comprise molecular grade pure water,or alternatively, a buffer solution, including but not limited to asolution of TRIS/EDTA; TRIS/acetate/EDTA, for example 4 mM Tris-acetate(pH 7.8), 0.1 mM EDTA, and 50 mM NaCl; TRIS/borate; TRIS/borate/EDTA;potassium phosphate/DMSO/glycerol; NaCl/TRIS/EDTA; NaCl/TRIS/EDTA/TWEEN;TRIS/NaCl/TWEEN; phosphate buffers; TRIS buffers; HEPES buffers; nucleicacid amplification buffers; nucleic acid hybridization buffers, etc.

Prior to forcing the elution fluid to flow through the component 122, anintermediate air-gap step may optionally be performed. A gas, preferablyair, may be forced to flow through component 122 after the wash solutionflows through and before the elution fluid flows through. The air-gapstep provides for clear separation of liquid phases, and helps at leastsubstantially dry the component 122 of any remaining wash solution priorto elution.

The component 122 is preferably heated as the elution fluid is forced toflow through it to increase elution efficiency. The heating ispreferably performed by supplying power to a resistive heating elementin a closed loop feedback system under the control of the processingelectronics in the cartridge. In the preferred embodiment, the component122 is heated to a temperature in the range of 60 to 95° C. as theelution fluid flows through the it.

Elution fluid containing the nucleic acid exits the component 122 andtravels down the channel 135 to the reagent chamber 141. The elutionfluid and nucleic acid contact and reconstitute dried PCR reagentscontained in the chamber 141, and the elution fluid, nucleic acid, andPCR reagents continue to flow into reaction chamber 143 for PCRamplification and detection. In an alternative embodiment, the elutionsolution already includes PCR reagents so that the reagent need not bedried in the chamber 141. Vents 145 in communication with the wastechamber 139 and the reaction chamber 143 allow release of gases duringthe process.

One advantage of the continuous-flow cartridge of the preferredembodiment is that it allows the analyte, e.g. nucleic acid, from arelatively large volume of fluid sample, e.g. several milliliters ormore, to be concentrated into a much smaller volume of elution fluid,e.g., 25 μL or less. In contrast to prior art devices, the cartridge ofthe present invention permits extraordinary concentration factors byefficiently extracting analyte from milliliter quantities of fluidsample and eluting the analyte into microliter quantity eluates. In thepreferred embodiment, the sample volume forced to flow through thecartridge is in the range of 1 to 100 mL, enabling concentration factorsof 100 or greater. For example, the analyte from 1 mL of fluid samplemay be captured in the device and concentrated into 10 μL or less ofelution fluid.

A fluid sample may be introduced into the cartridge by a variety ofmeans, manual or automated. For manual addition, a measured volume ofmaterial may be placed into a receiving area of the cartridge through aninput port and a cap is then placed over the port. Alternatively, agreater amount of sample material than required for the analysis can beadded to the cartridge and mechanisms within the cartridge can effectthe precise measuring and aliquoting of the sample needed for thespecified protocol.

It may be desirable to place certain samples, such as tissue biopsymaterial, soil, feces, exudates, and other complex material into anotherdevice or accessory and then place the secondary device or accessoryinto the cartridge causing a mechanical action which effects a functionsuch as mixing, dividing, or extraction. For example, a piece of tissuemay be placed into the lumen of a secondary device that serves as theinput port cap. When the cap is pressed into the port, the tissue isforced through a mesh that slices or otherwise divides the tissue.

For automated sample introduction, additional cartridge design featuresare employed and, in many cases, impart specimen accession functionalitydirectly into the cartridge. With certain samples, such as thosepresenting a risk of hazard to the operator or the environment, such ashuman retrovirus pathogens, the transfer of the sample to the cartridgemay pose a risk. Thus, in one embodiment, a syringe may be integratedinto a device to provide a means for moving external fluidic samplesdirectly into the cartridge. Alternatively, a venous puncture needle andan evacuated blood tube can be attached to the cartridge forming anassembly that can be used to acquire a sample of blood. Aftercollection, the tube and needle are removed and discarded, and thecartridge is then placed in an instrument to effect processing. Theadvantage of such an approach is that the operator or the environment isnot exposed to pathogens.

The input port can be designed with a consideration of appropriate humanfactors as a function of the nature of the intended specimen. Forexample, respiratory specimens may be acquired from the lowerrespiratory tract as expectorants from coughing, or as swab or brushsamples from the back of the throat or the nares. In the former case,the input port can be designed to allow the patient to cough directlyinto the cartridge or to otherwise facilitate spitting of theexpectorated sample into the cartridge. For brush or swab specimens, thespecimen is placed into the input port where features of the port andclosure facilitate the breaking off and retaining of the end of the swabor brush in the cartridge receiving area.

In another embodiment, the cartridge includes input and output tubesthat may be positioned in a sample pool of very large volume, such as aflowing stream of water, so that the sample material flows through thecartridge. Alternatively, a hydrophilic wicking material can serve as aninteractive region so that the entire cartridge can be immersed directlyinto the specimen, and a sufficient amount of specimen is absorbed intothe wicking material. The cartridge is then removed, and can betransported to the laboratory or analyzed directly using a portableinstrument. In another embodiment, tubing can be utilized so that oneend of the tube is in direct communication with the cartridge to providea fluidic interface with at least one interactive region and the otherend is accessible to the external environment to serve as a'receiver forsample. The tube can then be placed into a specimen and serve as asipper.

The cartridge itself may also serve as the actual specimen collectiondevice, thereby reducing handling and inconvenience. In the case ofspecimens involved in legal disputes or criminal investigations, thedirect accessing of the test material into the fluidic cartridge isadvantageous because the chain of custody is conveniently and reliablypreserved.

In general applications of the cartridge, chemical interactions of thefluid sample with one or more reagents may be required, so it isdesirable to include interactive regions that provide for chemicalreagents, the number and type depending on the specific analyticalprotocol to be facilitated. Multiple interactive regions, eachcontaining different reagents, can be arranged in series to enable thesequential processing of the sample.

Reagents may be exogenously introduced into the cartridge before use,e.g., through sealable openings in each region of the cartridge.Alternatively, the reagents may be placed in the cartridge duringmanufacture. The reagents may be disposed within the interactive regionsthat perform the operations for which the reagents will be used, orwithin regions leading to a particular interactive region.Alternatively, the reagents may be disposed within storage chambers influid communication with interactive regions.

The type of reagent utilized at an interactive region depends, interalia, on the fluid characteristics and size of the sample, the natureand concentration of the target constituents, and the desired processingprotocol. In the case of solution phase interactions, the reagents maybe aqueous solutions or dried reagents requiring reconstitution. Theparticular format is selected based on a variety of parameters,including whether the interaction is solution-phase or solid-phase, theinherent thermal stability of the reagent, speed of reconstitution, andreaction kinetics.

Liquid reagents may include, but are not limited to, buffer solutionssuch as saline, TRIS, acids, bases, detergent solutions, and chaotropicsolutions, which are commonly used for DNA and RNA purification andwashing. Dried reagents can be employed as precursor materials forreconstitution and solution-phase interaction or as solid-phasereagents, including pH indicators; redox indicators; enzymes such ashorseradish peroxidase, alkaline phosphatase, reverse transciptase, DNApolymerase, and restriction enzymes; enzyme substrates; enzyme-antibodyor enzyme-antigen conjugates; DNA primers and probes; buffer salts; anddetergents. Furthermore, solid-phase reagent coatings such as serumalbumin, streptavidin, and a variety of cross-linkable proteins such aspolysaccharides may be employed at the interactive region.

Dried reagents may also be contained within a membrane material that canbe employed as an interactive region by physical incorporation of thematerial into a region communication with fluidic channels. Cellulose,nitrocellulose, polycarbonate, nylon, and other materials commonly usedas membrane materials can be made to contain reagents. Such membranesare designed to capture target cells, effect lysis of host cells,release target nucleic acids, and separate contaminants that mayinterfere with the polymerase chain reaction or other analytical events.These papers may be positioned within a region to enable cross-flow ortangential flow of fluids. Because the papers can simultaneouslyphysically entrap target cells, lyse cells, and bind either targetanalytes or competing contaminants or analytical reaction inhibitors,they provide for multiple modes of activity at a single interactiveregion within the cartridge.

Reagents can be contained as liquids within specific regions of thecartridge; using conventional pouching or packaging techniques, thedesigns of which are optimized to allow integration into the cartridge.Reagents containing compounds that are thermally unstable when insolution can be stabilized by drying using common techniques such aslyophilization. Additives, such as simple alcohol sugars,methylcelluloses, and bulking proteins may be added to the reagentbefore drying to increase stability or reconstitutability. For thesereagents, reagent activity is reconstituted by rehydration with thefluid sample or with a separate reconstitution fluid, either bypre-mixing or preferably during sample flow.

A variety of techniques may be employed which provide for solid reagentdeposition patterns that facilitate uniform reconstitution. The reagentmay be deposited in a parabolic pattern mirroring the flow pattern ofthe fluid front in a wide, narrow channel, thereby increasing thelikelihood of uniform exposure of the sample contents to the reagent.The selection of sheets of dried reagents, layers of reagents, orindividual spot arrays depends on the desired reconstitution event, therate of reconstitution, and on whether additional mixing is employed.

For reagent spot arrays, ink-jet printing and piezocoupled micropipettetips can dispense drops of liquid reagent in a variety of uniform ornon-uniform patterns on the surface of an active region, and depositionof separate reagents in separate areas of the active region can beachieved if sequential modification of the fluid sample is desired, orif combined reagents cannot be dried as a single reagent. If the activeregion is a high surface-to-volume ratio structure, the region may bedipped into, sprayed with, or otherwise exposed tog a reagent, and driedbefore incorporation into the cartridge.

The operations enabled by specific chemical interactions includespecimen volume dilution; pH adjustment; biochemical solubilization;molecular aggregation; cellular or viral lysis; agglutination of targetcells or capture-particles; filtration; neutralization; specific analyteextraction and purification; contaminant extraction and separation;precipitation of specific molecules; binding of analyte to reportermoieties; and dried reagent reconstitution.

The overall geometry of the cartridge may take a number of forms. Forexample, the cartridge may incorporate a plurality of interactiveregions, e.g. channels or chambers, and storage regions, arranged inseries, so that a fluid sample is moved serially through the regions,and the respective operations performed in these regions. Alternatively,the cartridge may incorporate a central fluid interactive regionconnected to peripheral reagent or diluent storage chambers.

Generally, a single cartridge includes at least two distinct interactiveregions, and preferably, at least three or more distinct interactiveregions. Individual regions and regions may vary in size and shapeaccording to the specific function of the region or region. In somecases, elongated or spherical interactive regions or chambers may beemployed. In general, the interactive regions may vary in dimensionsfrom microscale (microns) to mesoscale (submillimeters) to macroscale(millimeters).

In some cases, a separate region may be used as a volumetric region,e.g., to precisely measure fluid volumes for introduction into anadjacent region. In such cases, the volume of the region is dictated byvolumetric needs of a given reaction. Further, the cartridge may befabricated to include a series of regions having varied dimensions andvolumes in comparison to each other.

Cross-sectional areas of the regions dictate the fluid resistance,pressure, and volumetric flow rates. The regions have dimensions orproperties (e.g., internal diameter, surface friction, materials,embedded chips, temperature, or other factors) that precisely controlthe volumetric flow rate, dwell times in the regions, processingefficiencies of on-board, pre-packaged reagents, and efficiencies ofsensors and detectors. Consequently, precise dwell times, reagentreconstitution rates, flow rates, flow directions, and all of theflow-through elements and parameters may be implemented.

The cartridge may be fabricated using one or more of a variety ofmethods and materials suitable for microfabrication techniques. Forexample, in preferred aspects, the cartridge may comprise a number ofplanar members that may individually be sheets or injection molded partsfabricated from a variety of polymeric materials, or may be silicon,glass, or the like. In the case of substrates like silica, glass orsilicon, methods for etching, milling, drilling, etc., may be used toproduce wells and depressions which make up the various regions,chambers and fluid channels within the cartridge capable of receivinginserts such as pouches, chips, papers, beads, gels, porous materials,tablets, and the like.

Microfabrication techniques, such as those regularly used in thesemiconductor and microelectronics industries, are particularly suitedto these materials and methods. These techniques include, e.g.,electrodeposition, low-pressure vapor deposition, glass bonding,photolithography, wet chemical etching, reactive ion etching (RIE),laser drilling, and the like. Where these methods are used, it willgenerally be desirable to fabricate the planar members of the cartridgefrom materials similar to those used in the semiconductor industry,i.e., silica glass, silicon, gallium arsenide, polyimides, metal filmsand the like. In additional embodiments, the cartridge may comprise acombination of materials and manufacturing techniques described above.In some cases, the cartridge may include some parts of injection moldedplastics, and the like, while other portions of the body may compriseetched glass or silicon members, and the like.

The cartridge may also incorporate one or more filters for capturingsample components, e.g., cells, spores, or microorganisms to be lysed.The filters may also be used for removing particulates, cell debris, andprotein solids from the sample. The filters may be within any region,e.g., within the fluid passages or channels leading between regions orwithin a particular interactive region. A variety of filter media may beused, including, e.g., cellulose, nitrocellulose, polysulfone, nylon,vinyl copolymers, glass fiber, micromachined structures, and the like.Similarly, separation media, e.g., ion exchange resins, affinity resinsor the like, may be included within the cartridge.

The surfaces of the fluid interactive regions that contact the fluidsample and reagents may be made hydrophobic or hydrophilic dependingupon the particular application. Where reagents involved in a particularanalysis are incompatible with the materials used to manufacture thecartridge, e.g., silicon, glass or polymeric parts, a variety ofcoatings may be applied to the surfaces of these parts that contact thereagents. For example, components that have silicon elements may becoated with a silicon nitride layer or a metallic layer of, e.g., goldor nickel, sputtered or plated on the surface to avoid adverse reactionswith these reagents.

Similarly, inert polymer coatings, Parylene® coatings, or surfacesilanation modifications may also be applied to internal surfaces of thecartridge in order to make the overall system more compatible with thereactions being carried out. For example, in the case of nucleic acidanalysis, it may be desirable to coat the surfaces with, e.g., anon-stick coating to prevent adhesion of nucleic acids to the surface.Additionally, patterned metal electrical conductors for activatingactuators, heaters, sensors, and the like may be used. Such conductorsmay be coated with insulator coatings in those instances whereelectrical leads are placed in contact with fluids, to prevent shortingout or gas formation from electrolysis. Such insulators are well knownin the art, e.g. screen-printed polymers, epoxies, ceramics and thelike.

Although the preferred embodiment incorporates flow controllers, e.g.valves, it is possible for a continuously-flowing fluid stream to beguided, divided and diverted to various regions within the cartridgewithout the incorporation of valves. In one embodiment, the fluid streamflows down a channel with relatively little flow resistance into asecond region, e.g., a waste chamber. The waste chamber may be ventedthrough a port blocked with a hydrophobic porous membrane, such asGoretex®. When the waste chamber is filled, and all the air in the wastechamber is expelled through the membrane vent, the fluid sample cannotpass through the membrane, and a back-pressure is developed.

The back-pressure is sufficiently large to force the remaining fluidstream through a smaller, secondary, capillary channel, pressuresensitive filter, or other flow restrictor located upstream from thefirst chamber. Once fluid flow is initiated through the small channel,no additional fluid will flow into the first channel and the fluidstream will be completely diverted into the secondary channel.Optionally, the smaller channel may be locally heated to inducediversion of the flowing sample into the smaller channel before thelarger region or chamber is full.

In addition, fluid may be prevented from flowing back upstream by fluiddiodes. FIG. 4 shows one example of such a fluid diode. Fluid ispermitted to flow in a direction from A to B, but prevented from flowingin the opposite direction from B to A. The diode 91 comprises a topportion 43 having a port 45 and an adjoining recess 47; a flex circuitplate 49 having a flap 51; and a bottom portion 53 having a channel 55.When the diode 91 is deactivated, a magnetic disc 57 on the flap 51 isattracted towards the top portion 43 by an external magnetic forceprovided by, e.g., the external instrument. The flap is biased againstthe recess 47 in the top portion 43, thus allowing fluid flowing throughthe port 45 to pass beneath the flap 51 and through the channel 55 ofthe bottom portion 53.

When the diode 91 is activated, the magnetic force is disabled and theflap 51 returns to a sealing position due to the spring constant of theflap which prevents fluid from passing from the port 45 beneath the flap51 and through the channel 55. In this manner, fluid present in thebottom portion 53 is prevented from flowing backwards through the port45 of the top portion.

The cartridge preferably has a venting element to release back pressureof fluids. The vent may include an opening to the external environment(e.g. inlet port or outlet port with or without a hydrophobic vent).Conveniently, the vent may be an internal expandable cavity such as acorrugated membrane or an elastic latex membrane. Release of fluidsthrough the vent may be passive or active, as in the application of avacuum to a port on the cartridge.

The inclusion of gas permeable fluid barriers, e.g., poorly wettingfilter plugs or hydrophobic membranes, in the cartridges also may beused to create a fluid direction and control system. Such filter plugs,when incorporated at the end of a chemically interactive region oppositea fluid inlet, allow air or other gas present in the interactive regionto be expelled during introduction of the liquid component into theregion. Upon filling of the region, the fluid sample contacts thehydrophobic plug thus stopping net liquid flow. Fluidic resistance mayalso be employed as a gas permeable barrier to accomplish this sameresult, e.g., using fluid passages that are sufficiently narrow toprovide an excessive resistance, thereby effectively stopping orretarding liquid flow while permitting air or gas flow.

A variety of materials are suitable for use as poorly wetting or gaspermeable filter plugs including, e.g., porous hydrophobic polymermaterials, such as spun fibers of acrylic, polycarbonate, Teflon®,pressed polypropylene fibers, or any number of commercially availablefilter plugs. Alternatively, a hydrophobic membrane can be bonded over athrough-hole to supply a similar structure. Modified acrylic copolymermembranes are commercially available from, e.g., Gelman Sciences (AnnArbor, Mich.) and particle-track etched polycarbonate membrane's areavailable from Poretics, Inc. (Livermore, Calif.). Venting of heatedchambers may incorporate barriers to evaporation of the sample, e.g., areflux chamber. Excessive evaporation of fluid from the sample may beprevented by disposing a mineral oil layer within the chamber and overthe top surface of the sample to permit the evolution of gas whilecontrolling evaporation. Lysing regions within the cartridge can bedesigned to effect lysing of target cells by physical, chemical or othermeans, or a combination of such means. Physical means includes themechanical disruption of the cells, such as by the vibration of glass orplastic beads or other particles or by impacting the target cells orviruses onto sharp microstructures. Thermal energy transfer, such as byheating a virus suspension to 95° C. or by repeated freeze-thawing ofactivated bacterial spores to disrupt cell walls, may also be used.

Chemical lysing can be employed alone or in combination with physical orultrasonic lysing. Typical chemical lysing agents fall into severalcategories, such as enzymes, detergents, and chaotropes. Lysosyme is anenzyme that hydrolytically attacks the cell walls of many bacteria;trypsin is a protease enzyme that breaks the cell membrane of mosteukaryotic cells. Other proteases with specificity for certain peptidesequences can be employed and are preferred if the target moiety isliable to certain proteases. Proteinase K is often used because it alsodigests nuclear proteins and host cell enzymes that may interfere withpolymerase chain reaction (PCR). For eucaryotic cells, detergents suchas Triton X-100 or sodium dodecyl sulfate solubilize the cell membraneand release intracellular contents. Chaotropes such as guanidineisothiocyanate or urea can be used to lyse cells and have the additionalbenefit of inhibiting RNAses that can destroy target RNA.

The mechanical disruption of target cells or viruses can be accomplishedwith interactive regions designed to tear the surface membrane or cellwall of the target organism via shearing or vibration. Vibration can beaccomplished by containing glass or other beads in a chamber, and bycoupling to the chamber a piezomembrane also incorporated into thecartridge. Alternatively, an ultrasonic transducer, such as anultrasonic horn, may be coupled to a wall of the chamber to transferultrasonic energy to the cells. The frequency and amplitude of theultrasound is tuned to correspond with the resonant frequency of thetarget cells and optimized to effect lysis with minimal heating orcavitation, though the latter may be required for efficient lysis.

Microfabricated chips can be designed to effect one or more modes ofphysical or chemical disruption of host cell walls or membranes. In oneembodiment, the chip has an integral heater and high surface areamicrostructures derivatized with amino-silane to allow the chemicalconjugation of antibodies with specificity and avidity for the surfaceproteins of a target cell or virus. When a fluid sample containing thetarget cell or virus flows through the chip, the target cell or virus isbound by antibodies linked to the high-surface area microstructures andremoved from the flowing fluid stream. The microstructures are heated to95° C. at a later time causing the viruses to lyse.

Other methods of cell extraction may also be used, e.g., employing achannel with restricted cross-sectional dimensions so that shear stresscauses cell lysis when the sample is passed through the channel atsufficiently high pressure. Alternatively, cell extraction anddenaturing of contaminating proteins may be carried out by applying analternating electrical current to the sample. Numerous other methods maybe utilized within the cartridge to effect lysis and extraction.

Following extraction, it will often be desirable to separate the nucleicacids from other elements of the crude extract, e.g., denaturedproteins, cell membrane particles, and salts. Removal of particulatematter is generally accomplished by filtration, flocculation and thelike. A variety of filter types may be readily incorporated into thechemically and/or mechanically interactive regions of the cartridge.Further, where chemical denaturing methods are used, it may be desirableto desalt the sample prior to proceeding to the next step. Desalting ofthe sample and isolation of the nucleic acid may be carried out, e.g.,by binding, the nucleic acids to a solid phase and washing away thecontaminating salts, or by performing gel filtration chromatography onthe sample, by passing salts through dialysis membranes, and the like.Suitable solid supports for nucleic acid binding include, e.g., filters,beads, fibers, membranes, glass wool, filter paper, polymers, and gelexclusion media.

In some embodiments, enzymes, such as a polymerase enzyme, may bepresent within an amplification region, coupled to a suitable solidsupport, or to the walls and surfaces of the region. Suitable solidsupports include those that are well known in the art, e.g., agarose,cellulose, silica, divinylbenzene, polystyrene, etc. Coupling of enzymesto solid supports has been reported to impart stability to the enzyme inquestion, which allows for storage of days, weeks or even months withouta substantial loss in enzyme activity, and without the necessity oflyophilizing the enzyme. Monoclonal antibodies are available which bindthe enzyme without affecting its polymerase activity. Consequently,covalent attachment of the active polymerase enzyme to a solid support,or the walls of the amplification region may be carried out by using theantibody as a linker between the enzyme and the support.

In another aspect of the invention, ligand binding methods can be usedin the cartridges for binding and capturing specific cell types and/orother analytes. Ligand binding entities, such as nucleic acids andproteins, may be located at selected capture regions, attached to thesurface(s) of the analyte capture components, to form a specificanalyte-reacting region. Ligand coupling chemistries, such assilane-based chemistries, may be used. Homo- or hetero-bifunctionallinkers, with one functionality binding to the internal surface and theother to a target in the test sample, may be employed. A samplecontaining the target analyte is passed continuously through thecartridge and the analyte binds to the ligand covered surface. Aftersubsequent washing with one or more wash solutions, the ligand-analytecomplexes can be eluted. Alternatively, a secondary anti-analytemolecule conjugated to a reporter molecule may be passed through thecartridge, so that the conjugate is captured by the analyte. Thiscomplex may also be eluted.

In particularly preferred embodiments, the cartridge is made from atleast one injection molded, press molded or machined polymeric part thathas one or more wells or depressions manufactured into its surface todefine several of the walls of the interactive regions. Examples ofsuitable polymers for injection molding or machining, include, e.g.,polycarbonate, polystyrene, polypropylene, polyethylene, acrylic, andcommercial polymers such as Kapton®, Valox®, Teflon®, ABS, Delrin® andthe like. A second part that is complementary in shape is mated to thesurface of the first part to define the remaining wall of the cartridge.The mating part or a third part may be a printed circuit board forimplementing electrical contact directly with the fluid or indirectlyvia the cartridge.

The cartridge may be fabricated in such a way that specific regions orregions interact with an external instrument via exchange ofelectromagnetic radiation. Many plastics commonly used for suchcartridges (e.g. polypropylene and polycarbonate) are opticallytransparent. In general, insulating materials allow electromagneticradiation to pass over a wide frequency range. Such radiation may be ofany frequency consistent with the intended application. For example,radio waves may be used as an alternative means of communicating withthe cartridge. Radio waves may also be used to supply small amounts ofpower to any internal circuitry within the cartridge. Microwavefrequencies may be used to induce heating of the fluid sample. Infraredsignals may be used for heating, or for data exchange via an IR link,similar to those used in personal computers.

Optical frequencies, using light emitting diodes (LEDs) andphotodetectors, such as photodiodes, are useful for detection of fluidpresence (by detecting changes in optical transmittance), and formonitoring of chemical reactions (by, e.g., measuring absorption,fluorescence, or luminescence at specific wave lengths). Optical, aswell as ultraviolet frequencies may be used to excite fluorescence ofreaction products for detection. These frequencies may also be used toinduce or accelerate chemical reactions.

Higher frequency radiation, such as deep UV or x-ray radiation, is alsopossible for specific applications, although the sources for these typesof radiation may not always be practical in a small instrument. Sourcesof ionizing radiation (such as radioactive materials) could bereasonably incorporated into an instrument, and such radiation used forspecific purposes within the cartridge, such as the enhancement ofreactions or detection of specific fluid components or properties.

The cartridge may be fabricated in such a way that specific regions orregions may interact with the external environment via magnetic forces.For example, a region of the cartridge may contain a reservoir ofmagnetic beads. Such beads can be functionalized with various bindingagents. By applying a series of magnetic fields to the cartridge (e.g.by means of switchable electromagnets) these beads may be vibrated ormoved from one region to another. Using AC electromagnetic fields, suchbeads may be caused to circulate within a small region of the cartridgeto mix fluids within the cartridge.

Magnetic forces may also be used to operate small valves within thecartridge for fluid control. A small strip of magnetic material may beincorporated into the cartridge to divert the fluid flow along oneparticular flow path. Another possibility is to fabricate the magneticstrip in such a way that it returns to the first position when the fieldis removed. The strip could be fabricated in such a way as to bemechanically bistable. Application of a magnetic pulse to the stripcauses a mechanical transition from the initial bistable state to thesecond state. In this second state, the strip diverts the fluid flow toan alternative path. An array of such valves allows complete control ofthe fluid motion.

The cartridge may be fabricated so that specific regions may interactwith the external instrument via electric fields. By fabricating verythin regions within the cartridge, and by mating these withcorresponding conductive areas within the instrument, electric fieldsmay be applied to the fluid without the need for any electricalconnections to the cartridge itself. Such electric fields may be used tomove charged molecules from one surface to the other within thecartridge. By proper design of the fluidic paths, such a configurationmay be used to separate charged from uncharged molecules, or to attractand hold charged molecules while other unwanted molecules are flushedfrom the system.

A number of the operations performed by the various interactive regionsof the cartridge require a controllable-temperature. For example, PCRamplification requires cycling of the sample among a strand separationtemperature, an annealing reaction temperature; and an extensionreaction temperature. A number of other reactions, including isothermalDNA amplification techniques, ligand binding, enzymatic reactions,extension, transcription and hybridization reactions are also generallycarried out at optimized, controlled temperatures.

Temperature control is generally supplied by resistive heaters which areprepared using methods that are well known in the art. For example,these heaters may be fabricated from thin metal films applied within oradjacent to channels or chambers using well known methods such assputtering, controlled vapor deposition, screen printing and the like.The heater is electrically connected to a power source which delivers acurrent across the heater. The electrical connections may be fabricatedusing methods similar to those described for the heaters.

In one embodiment, a controllable heater is disposed within or adjacentto a region for thermal control of the sample. Thermal control iscarried out by varying the current supplied to the heater to achieve thedesired temperature for the particular stage of the reaction.Alternatively, thermal control may be achieved by transferring the fluidsample among a number of different reaction regions or regions of thesame cartridge, having different, constant temperatures, or by flowingthe sample through a serpentine channel which travels through a numberof varied temperature zones. Heating may alternatively be supplied byexposing the region to a laser or other radiation source.

Resistive heater elements may also be incorporated into regions of thecartridges by diffusing silicon into the regions or by depositingthin-film metal, carbon, or polysilicon at selected regions. Controlledheating provides additional functional capabilities, such as mixing,dissolution of solid reagents, lysing, thermal denaturation of proteinsand nucleic acids and lysis of cells, elution of bound molecules,enhanced diffusion rates of molecules in the sample, and modification ofsurface binding coefficients, as well as high efficiency thermal cyclingfor polymerase and ligase chain reactions. Cooling features may also beexploited in high surface area regions, for example, with externalcooling fins.

Preferably, the heaters are capable of producing temperatures in excessof 100° C. without suffering adverse effects as a result of the heating.The heaters may be provided as a layer on one surface of an interactiveregion or other region, or may be provided as molded or machined insertsfor incorporation into a region or region. Control of the power sourceis typically carried out by an appropriately programmed processor, suchas the processor in the external instrument. The heaters may beincorporated within the cartridge by depositing a resistive conductivefilm or insert on a surface of the cartridge, or alternatively, theheaters may be provided externally, e.g. in the instrument, and appliedto the exterior of the cartridge, adjacent to a particular region, sothat heat is conducted into the region.

Temperature controlled regions may also include miniature temperaturesensors for monitoring temperatures and thereby controlling theapplication of current across the heater. A wide variety of microsensorsare available for determining temperatures, including, e.g.,thermocouples having a'bimetallic junction which produces a temperaturedependent electromotive force (EMF), resistance thermometers whichinclude material having an electrical resistance proportional to thetemperature of the material, thermistors, IC temperature sensors, quartzthermometers and the like. Alternatively, the temperature coefficient ofresistance of the heater itself may be monitored to control the heatinput.

The temperature measured by the temperature sensor and the input for thepower source will typically be input to a processor, e.g. amicroprocessor or microcontroller in the external instrument, which isprogrammed to receive and record this data. The same processor willtypically include programming for instructing the delivery ofappropriate current for raising and lowering the temperature of theinteractive region or regions. For example, the processor may beprogrammed to take the interactive region through any number ofpredetermined time/temperature profiles, e.g., thermal cycling for PCR,and the like. Given the small size of the cartridges of the invention,cooling of an interactive region will typically occur through exposureto ambient temperature. However, additional cooling elements may beincluded if desired, e.g., coolant systems, Peltier coolers, waterbaths, heat pipes, and the like.

In alternate aspects, mixing may be accomplished by the incorporation offerromagnetic elements within the cartridge which may be vibrated bysupplying an alternating current to a coil adjacent the device. Theoscillating current creates an oscillating magnetic field through thecenter of the coil which results in vibratory motion and rotation of themagnetic particles in the cartridge and mixing of the fluid components.In addition to sensors for monitoring temperature, the cartridge maycontain sensors to monitor the progress of one or more of the operationsof the device. For example, optical sensors and pressure sensors may beincorporated into one or more regions to monitor the progress of thevarious reactions, or within flow channels to monitor the progress offluids or detect characteristics of the fluids, e.g., pH, temperature,electrical conductance, capacitance, fluorescence, viscosity,(chemi)luminescence, color, and the like.

The cartridge will typically include temperature sensors andcontrollers. For example, a heating element or temperature control blockmay be disposed adjacent the external surface of a chemicallyinteractive region to transfer heat to the region. In this case,preferred cartridges include a thin external wall for regions in whichthermal control is desired. This thin wall may be a thin cover element,e.g.; polycarbonate sheet, or high temperature tape, i.e. siliconeadhesive on Kapton® tape (commercially available from, e.g., 3M Corp).In one embodiment, the cartridge may comprise two or more componentsthat are fabricated separately, and then bonded together. Some surfacesof the components ultimately become the interior of the fluid flowregions or channels.

On such surfaces, conductive layers may be deposited. These could be oneof several metals, for example gold, chrome, platinum, silver, carbon,copper or other metals, deposited by standard thin film depositiontechniques such as plating, evaporation or sputtering. Another methodfor deposition of such conductive materials is via thick filmtechnology. In this method, conductive pastes or inks are deposited byscreen printing, and then baked to drive off solvents and leave behindthe final conductor. Finally, thin films of carbon are commonly used forlow cost conductive materials. These can also be screen printed andbaked at low temperatures to form conductive layers.

Any of the above methods are useful for allowing conduction ofelectrical signals from the external environment, through the fluid sealarea, and into the interior of the cartridge. These conductors can bemade very thin, limited only by the necessary conductivity. In the caseof a cartridge, the thickness of the conductors may be on the order of0.0254 mm.

Electrical signals through such conductors may be used in a number ofways, both as inputs to the cartridge and as outputs from it. Somesignals involve making a circuit, part of which is the fluid itselfwithin the cartridge. In one embodiment, such a circuit is used simplyto sense the presence or absence of the fluid. Two conductive terminalsare routed to regions within the fluid channel, close to one another butnot connected to each other. External electronics monitors the impedancebetween these conductors, by, for example, applying a small voltagebetween them and monitoring the current flow. When no fluid is present,the impedance will be very high. However, when fluid passes this pointin the channel, the fluid will bridge the gap between the two terminals.Since the fluids typically used in biological and chemical applicationsare at least mildly conductive, this fluid will cause the impedance inthe circuit to decrease dramatically. This decrease in impedance can besensed by the electronics, and decisions made based on this input. Byplacing several such circuits along the length of any fluid channel, theexternal electronics may be used to monitor the fluid velocity, thusmonitoring the progress of the intended fluidic processing.

Electrodes in contact with the fluid might also be used for monitoringspecific characteristics of the fluid. Capacitance, fluid conductivity,fluid pH, capacitance, reaction region humidity (e.g. in paper basedcartridges) are all examples of specific fluid parameters that might bemonitored by electronic means. Specific electrode configurations arealso possible to allow electrochemical detection of reaction products.

Another example is the use of such electrical connections into the fluidfor manipulation of biomolecules such as DNA. Such molecules can bemoved through fluids by DC electrophoresis. In this case, one electrodemakes contact with the fluid as a counter electrode. Many otherelectrodes can be biased with respect to the counter electrode toattract charged molecules. For example, some macromolecules such as DNAare negatively charged. By biasing electrodes positively with respect tothe counter electrode, these macromolecules can be attracted to thepositive electrodes. This may be useful for isolating such moleculesfrom other fluidic components, or for attracting such molecules tospecific reaction regions within the cartridge.

Another electronic technique useful for movement and isolation ofbiomolecules is AC dielectrophoresis. In this case, two or moreelectrodes are typically configured close to one another, and in aphysical configuration which yields non-uniform electric fields. ACfields at frequencies up to tens of MHz are known to induce electricalpolarization of such molecules, causing them to move, or be attractedto, regions where they may be isolated or further processed. Moleculesalso have unique signatures, i.e. particular molecules respond to aparticular frequency of excitation. Thus specific molecules can beisolated from the fluidic sample by tuning of the frequency of the ACexcitation. By using traveling wave excitation along a series ofelectrodes, these specific molecules can be moved from place to place.

Another application of an electrical connection is that of driving anelectrolysis reaction to realize fluid movement. Electrical connectionsto a fluid reservoir could be used to realize an electrolytic pump(e-pump). In such a device, current is passed through a reservoir ofelectrolyte. This current causes gas evolution as the electrolytesolvent is decomposed into gases such as oxygen and hydrogen. Thesegases build up localized pressure and can serve as a motive source. Thispressure can be transmitted to the process fluid within the cartridgethrough, e.g. a flexible membrane, thus realizing fluid motion of thefluid to be processed.

FIGS. 5A and 5B show one such electrolytic pump 25. As shown in the planview of FIG. 5A, the pump 25 includes electrodes 27 having a star shapeto assure that a current path is always available even after bubblesbegin to form inside of the reservoir 29. A sealing ring 4 entrapselectrolyte within the reservoir 29. As shown in the schematic side viewof FIG. 5B, fluid 39 is contained within a pouch 35 having an expandablemembrane 37. The fluid contacts electrodes 27 and decomposes whenelectric current is applied to the electrodes. The decomposing fluidcreates a pressure build-up within the pouch 35. As the pouch expandsdue to increased pressure, the pouch biases against a liquid reagentpouch (not shown), thus forcing the liquid reagent contained within theliquid pouch to be released. By controlling the current (power) to theelectrodes 27, and in conjunction with the aforementioned means formonitoring of fluid flow velocity, a closed loop fluid flow controlsystem can be realized. Such an implementation opens up manypossibilities for very well controlled reactions, as the fluid velocity(and hence residence times at various reaction regions), at variouspoints in the processing cycle can be independently controlled andmonitored.

FIG. 6 shows a schematic, cross sectional view of a preferredmicrofabricated chip 20 to be used as the flow through component in thecartridge of FIG. 2. The chip 20 is used to capture a desired analyte,e.g. nucleic acid, from a fluid sample and to provide a highlyconcentrated eluate of the analyte. The chip 20 includes a body havingformed therein an inlet port 28, an outlet port 30, and an extractionchamber 26 for extracting the analyte from the fluid sample as the fluidsample flows through the body. The chamber 26 is in fluid communicationwith the inlet and outlet ports 28 and 30, and the ports are preferablypositioned on opposite sides of the chamber 26 to permit continuousfluid flow through the chamber.

The body preferably comprises a base substrate 22 and a top substrate 24bonded to the base substrate 22. The substrates 22 and 24 may compriseany suitable substrate materials, such as silicon, glass, silicondioxide, plastics, or ceramics. In the preferred embodiment, the chamber26 is formed in the base substrate 22, and the fluid ports 28 and 30 areformed in the top substrate 24. In alternative embodiments, however,many different configurations are possible, e.g., the chamber 26 may bepartially or completely formed in the top substrate 24, the fluid portsmay be formed in bottom or sides of the base substrate 22, etc. Severalof these alternative embodiments will be described below.

The chamber 26 has internal attachment surfaces having sufficiently highsurface area and binding affinity with the target analyte to capture theanalyte as the fluid sample flows through the chamber. In the preferredembodiment, the internal attachment surfaces are formed by an array ofinternal microstructures, preferably high aspect ratio columns 32,integrally formed with a wall of the chamber 26 and extending into thechamber. For simplicity of illustration, only twenty-five columns areshown in the schematic view of FIG. 6. It is to be understood, however,that the chip of the present invention may include many more columns. Ingeneral, it is preferred to fabricate the chip with at least 100columns, and more preferable to fabricate the chip with 1,000 to 10,000columns. The number of columns depends, inter alia, on the amount andconcentration of analyte in the sample, the dimensions of the chamber,the spacing of the columns, the flow rate of fluid through the chamber,etc. Specific techniques for fabricating the chip are described below.

FIG. 8 shows a portion of the array of columns 32 extending from abottom wall 23 of the extraction chamber. The columns 32 preferably havean aspect ratio (ratio of height to width or diameter) of at least 2:1,and more preferably have an aspect ratio of at least 4:1. The highaspect ratio columns 32 provide a large surface area for capturing theanalyte. As the fluid sample flows through the chamber, the analytecontacts and adheres to the surfaces of the columns 32. To elute theanalyte, an elution fluid is forced to flow through the chamber,releasing the analyte, e.g. nucleic acid, from the surfaces of thecolumns 32 into the elution fluid. In the preferred embodiment, thecolumns 32 have a height equal to the depth of the extraction chamber,preferably at least 100 μm. In alternative embodiments, the extractionchamber may have a shallower depth, but depths of less than 100 μm maycause excessively slow fluid flow through the chamber.

FIG. 9 shows a schematic view of the array of columns 32 disposed in thechamber 26. Fluid enters the chamber 26 through the inlet port 28 andflows between the columns 32 to the outlet port 30. The columns 32 arepreferably arranged in an array that optimizes fluid interaction withthe surfaces of the columns as the fluid flows through the chamber 26.The optimization of the column arrangement permits faster flow rates offluids through the chamber without losing efficiency of extraction.

In the preferred embodiment, the columns 32 are disposed in rows, witheach of the columns in a row spaced a uniform distance from adjacentcolumns in the row, i.e. the columns in a row preferably have uniformcenter to center spacing. For example, FIG. 9 illustrates ten horizontalrows of uniformly spaced columns 32. In addition, adjacent rows arepreferably offset from each other such that the columns in each row aremisaligned with the columns in an adjacent row. For example, each row ofcolumns in FIG. 9 is offset horizontally from an adjacent row.

Also in the preferred embodiment, the rows are offset such that thecolumns in each row are misaligned with the columns in at least twoprevious and/or successive rows. The misalignment may be in a pattern ofsuccessive rows, where the chamber includes one pattern or a repeatedpattern. For example, the pattern may repeat every three to ten rows. Inthe alternative, the misalignment of columns may be random from row torow.

Generally, any two adjacent rows in the array should not be offset fromeach other such that the columns in the first row are aligned exactlyhalfway between the columns in the second row. Instead, it is presentlypreferred to offset adjacent rows a distance greater than or less than50% of the center to center spacing between the columns. Thisarrangement provides for an asymmetrically split flow pattern throughthe chamber to ensure that each branch of the fluid stream interacts asstrongly as possible with the surfaces of the columns.

A specific example of a suitable arrangement of columns will now begiven with reference to FIG. 9. In each row, the center to centerspacing between adjacent columns is 15 μm. The columns are arranged in apattern that repeats every five rows. In particular, each of the topfive rows is offset 6 μm from a previous/and or successive row. Thebottom five rows (the sixth through tenth rows) repeat the pattern ofthe top five rows, with the sixth row being aligned with the top row,e.g., column 32A is aligned with column 32B. Of course, this is just oneexample of a suitable array of columns and is not intended to limit thescope of the invention. It will be apparent to one skilled in the artfrom this description that the columns may be arranged in many otherpatterns, preferably within the general guidelines set forth above.

FIG. 10 shows a top plan view of two adjacent columns 32 in a row. Thecolumns 32 preferably have a cross sectional shape and size whichmaximizes fluid contact with the surfaces of the columns while stillallowing for smooth fluid flow through the chamber. In the preferredembodiment, this is achieved by fabricating columns having a long andthin cross sectional shape, preferably a streamlined shape, such as thehexagonal shapes shown in FIG. 10. In particular, each column 32preferably has a ratio of cross sectional length L to cross sectionalwidth W of at least 2:1, and more preferably of at least 4:1. Further,the cross sectional length L is preferably in the range of 2 to 200 μm,and the cross sectional width W is preferably in the range of 0.2 to 20μm.

The gap distance S between adjacent columns in a row is preferablyselected to be as small as possible while still allowing fluid to flowbetween the columns without excessive resistance. In general, the gapdistance S may range from 0.2 to 200 μm, and more preferably, is in therange of 2 to 20 μm. The range of 2 to 20 μm is currently preferredbecause it provides for substantial fluid contact with the surfaces ofthe columns without causing excessive resistance to the fluid flowthrough the chamber. The center to center spacing C between adjacentcolumns in a row is the sum of the cross sectional width W and gapdistance S, and is preferably in the range of 2.0 to 40 μm.

The length of the extraction chamber 26, its vertical dimension in FIG.9, is preferably in the range of 100 to 5000 μm, and more preferably atleast 1000 μm. The width of the extraction chamber 26 is preferably inthe range of 100 to 3000 μm. The fluid ports 28 and 30 each preferablyhave a width or diameter of at least 100 μm. It is presently preferredthat the chamber 26 have a minimum length of 1000 μm to allow sufficientroom for the array of columns 32 and for the fluid ports 28 and 30. Inparticular, it is presently preferred to confine the array of columns 32to the center area of the chamber 26, leaving open space at the ends ofthe chamber 26 where the fluid ports 28 and 30 join the chamber. Thisarrangement increases uniformity of fluid flow into the chamber 26 priorto the fluid flowing between the columns 32.

Referring again to FIG. 6, the internal surfaces of the chamber 26, e.g.the columns 32 and chamber walls, may be coated with a substance havinga high binding affinity with the target analyte. Suitable substancesinclude, for example, silicon, silicon derivatives such as silicondioxide, polymers, polymer derivatives such as polyamides, nucleicacids, certain metals, polypeptides, proteins, and polysaccharides.

The silicate (SiO₂) nature of glass can attract and bind nucleic acids.Silicon, when it becomes oxidized, results in a similar surfacechemistry. Non-permanent (non-covalent) attachment (adsorption) to sucha surface is typically based on weak dipole, hydrogen bonding, or ionicinteractions between the surface and moiety to be captured. Theseinteractions are reversible via changes in the ionic nature of thesolvent and/or surface, heat, or other physiochemical means. Manymaterials can be tailored to have a variety of interactions withsolvents and solutes in solution. Polymers can have active surfacegroups that provide specific interactive forces, and they can havecopolymers or dopants that provide ionic or even hydrogen bindingcapabilities. Some polymers can have reversible polarities or adjustableconductivity. Synthetic and some natural polypeptides and proteins haveshown a similar capability to have a variety of interactions with solutemolecules. Metals, such as gold, are well known to have the ability tocapture DNA, and due to its electronic nature, can change the ionicinteractions with solutes.

The internal surfaces of the chamber 26 may also be coated with asubstance having a high binding affinity with a specifically targetedanalyte, e.g., a specific sequence of RNA from a virus or a specificsequence of DNA from a bacteria. This may be accomplished by coating theinternal surfaces with a specific nucleic acid sequence complementary tothe target nucleic acid sequence. The surfaces may be coated duringmanufacture of the chip or immediately prior to use.

The microfluidic chip 20 preferably includes a heater for heating theextraction chamber 26. The heater allows for highly efficient elution ofthe analyte from the chamber so that a large amount of analyte may bereleased into a small volume of elution fluid. The heater may also beused to facilitate capture of the analyte. One advantage of the use of aheater in a small volume microchamber is that minimal energy is requiredto heat the chip.

In general, the heater may comprise any suitable mechanism for heatingthe chamber 26, including resistive heaters, optical heaters fordirecting visible or infrared light, or electromagnetic heaters. If thebody of the chip 20 is fabricated from an electrically conductivematerial, preferably silicon, the heater may simply comprise a powersource and electrodes for applying a voltage across a portion of thebody forming the chamber 26. Also, high thermal conductivity of thematerial allows for fast heating times, reduced power requirements, andhighly uniform temperatures. This embodiment is described in greaterdetail below.

In the preferred embodiment, the heater comprises a resistive heatingelement 34 coupled to the bottom wall of the chamber 26. As shown inFIG. 7, the resistive heating element 34 is preferably a thin film ofmetal, carbon, or polysilicon that is patterned on the bottom surface ofthe substrate 22. Alternatively, the heating element may comprise alaminated heater source, such as an etched foil-heating element,attached to the substrate 22. Electrically conductive bond pads 38A and38B are also patterned on substrate 22 for electrically contactingopposite ends of the heating element 34.

The bond pads 38A and 38B may be connected by electrical leads to apower source for applying a voltage across the heating element 34.Control of the power source is preferably carried out by anappropriately programmed controller, such as a computer, microprocessor,or microcontroller in the cartridge or external instrument. Thecontroller may be programmed to take the chamber 26 through any numberof predetermined time/temperature profiles by varying the amount ofpower supplied to the heating element 34.

The microfluidic chip also preferably includes one or more temperaturesensors in communication with the controller for measuring thetemperature of the extraction chamber 26. In general, the temperaturesensor may be any suitable device for measuring temperature, such as athermocouple, resistance thermometer, thermistor, IC temperature sensor,quartz thermometer, or the like. Alternatively, the temperaturecoefficient of resistance of the heating element 34 may be utilized as ameans to monitor the chamber temperature and to control the heat inputby measuring the resistance as indicative of temperature.

In the preferred embodiment, the temperature sensor comprises a strip 36of electrically conductive material patterned on the substrate 22. Thestrip 36 comprises a material having an electrical resistance dependenton the temperature of the material, so that the temperature of thechamber 26 may be monitored by monitoring the resistance of the strip36. Electrically conductive bond pads 40A and 40B are also patterned onsubstrate 22 for electrically contacting opposite ends of the sensorstrip 36.

In an alternative embodiment, the substrate 22 may also have anadditional bond pad 42 patterned thereon for providing a bulk contact tothe substrate 22. The bulk contact may be used to charge the internalattachment surfaces of the chamber 26 with a voltage to attract and/orelute nucleic acid. Suitable metals for forming the resistive heatingelement, sensor strip, and bond pads include aluminum, gold, silver,copper, and tungsten.

The bond pads 40A and 40B are connected by electrical leads to thecontroller, and the controller is preferably programmed to adjust theamount of power supplied to the heating element 34 in dependence uponthe resistance of sensor strip 36. The controller, power source, heatingelement, and temperature sensor thus form a closed loop temperaturecontrol system for controlling the temperature of the chamber 26.Although a closed loop system is presently preferred, in alternativeembodiments the temperature sensor may be eliminated and the chip may beoperated in an open loop mode. Further, the processing electronics,including e.g., one or more microprocessors, multiplexers, power controlcircuitry, and sensor circuitry, may be included in the chip or locatedexternally to the body of the chip and connected thereto.

The microfluidic chip is preferably used in combination with acartridge, as previously described with reference to FIG. 2. Oneadvantage of the flow-through chip is that it allows the analyte from arelatively large volume of fluid sample, e.g. several milliliters ormore, to be concentrated into a much smaller volume of elution fluid,e.g., 25 μL or less. In particular, the ratio of the fluid sample volumeforced to flow through the device to the volume capacity of theextraction chamber is preferably at least 2:1, and more preferably atleast 10:1. In the preferred embodiment, the extraction chamber has avolume capacity in the range of 0.1 to 25 μL, and the volume of fluidsample forced to flow through the device is in the range of 1 to 100 mL,enabling concentration factors of 100 or greater.

Another advantage of the microfabricated chip is that it allows forrapid and direct heating of the internal attachment surfaces of thechamber. The integral nature and high thermal conductivity of thechamber walls and column structures allow for rapid heat transfer fromthe heating element directly to the attachment surfaces withoutnecessitating heating of the fluid in the chamber. This improvementinefficiency is significant in terms of the speed, precision, andaccuracy of the heating, as well as in the reduction in power requiredfor the heating. In particular, the rapid and direct heating of theinternal surfaces to which the analyte is bound greatly increases thedegree and efficiency of the elution, and provides a significantimprovement over prior art methods and devices.

A further advantage of the chip is that it includes an array ofintegrally formed microstructures, preferably high aspect ratio columns,which provide for a high degree of efficiency and control in separatinganalyte from a fluid sample. In addition to allowing direct and rapidheating of attachment surfaces, the microstructures greatly increase theeffective surface area of the chamber which may be used to capture andelute analyte.

Further, with regularly spaced columns, the diffusion distances betweenthe columns are consistent and there is uniformity of fluid flow so thatevery analyte is subjected to the same “micro-environment” as opposed tothe random nature of beads and fibers. This uniformity allows forpredictability of extraction parameters including the time required foreach processing step, flow rates, heating amounts, fluid volumes, etc.In addition, the increased efficiency obtained by using an array ofinternal microstructures and by rapidly and directly heating attachmentsurfaces allows for the efficient extraction and elution of analyteswith relatively high fluid flow rates through the chamber. Thisdecreases the overall time required for the extraction and elution.

The microfabricated chips of the present invention are also useful forcombinatorial synthesis of biopolymers such as oligonucleotides andpolypeptides. Combinatorial synthesis allows very large numbers ofsequences to be synthesized in a device by transporting, concentrating,and reacting monomers, coupling and deblocking reagents, and catalystsat separately addressable reaction/extraction microstructures. This useexploits the ability of the device to insulate selected microstructuresfrom each other and from nearby reagents.

The chip 20 may be fabricated using a variety of techniques, includingphotolithography and/or micromachining. Fabrication is preferablyCarried out on silicon or other suitable substrate materials such asglass, silicon dioxide, plastics, or ceramics. A preferred method forfabricating the microfluidic device using deep reactive ion etching(DRIE) will now be described.

A 100 mm, n-type (100), 0.1 to 0.2 ohm-cm, double side polished siliconwafer is used as starting material for the base substrate 22. The waferthickness is preferably in the range of 350 to 600 μm, depending on thedesired structure. In one embodiment of making the chip, an ohmiccontact may be made by using phosphorous ion implantation into, a regionin the backside, preferably to a depth of 0.2 to 5 μm. Alternatively, ap-type silicon wafer may be used, and the ohmic contact made using boronion implantation. Implantation is followed by heating of the substrateto activate the dopant.

The wafer is then spun with photoresist (commercially available from,e.g., Shipley) on the frontside to obtain a photoresist thicknesssufficient to mask the DRIE process. This thickness depends upon thefinal desired depth of the etch. The ratio of silicon etch rate tophotoresist erosion rate is typically greater than 50:1. To etchstructures that are 200 μm deep, 4 μm of photoresist is usuallysufficient. The photoresist is softbaked at 90° C. for about 30 minutes,then exposed with the desired mask pattern, developed, and hardbakedusing processes well known in the art of silicon wafer processing.

FIG. 11 illustrates a sample mask pattern on the frontside of the wafer.The etch mask defines a chamber pattern 44 for forming the extractionchamber in the substrate 22 and an array of column patterns 46 forforming a corresponding array of columns in the substrate. Due to spacelimitations in drawing size, the etch mask is illustrated with onlyseveral hundred column patterns 46. In the preferred embodiment,however, the array includes 1,000 to 10,000 column patterns for forminga corresponding number of columns in the substrate 22.

The patterned wafer is then etched using a DRIE process to form theextraction chamber and integral columns. The DRIE process involves theuse of inductively coupled plasma etching and deposition in analternating fashion, using fluorine based chemistry. Aspect ratios of20:1 in etched structures are easily realized. The etch rate istypically 2 μm/min or higher.

After etching, the remaining photoresist is removed from the wafer,e.g., by oxygen plasma etching or wet chemical stripping in sulfuricacid. The substrate is then oxidized to cover the internal surfaces ofthe chamber, i.e., the chamber walls and surfaces of the columns, withan oxide layer. The oxide layer is preferably 1 to 100 nm thick, and maybe formed using any well known technique, e.g., thermal growth, chemicalor electrochemical growth, or deposition.

An electrically conductive material, e.g., aluminum, gold, or copper, isthen deposited and patterned on the backside of the substrate to formthe resistive heating element, temperature sensor, and bond pads.Different materials may be used to form the heating element and sensor.Specific techniques for patterning metal on a substrate are well knownin the art. The substrate is then anodically bonded to a thin, e.g., 500μm, Pyrex™ glass cover. The glass cover has holes fabricated in it,e.g., by ultrasonic milling, which form the fluid ports to the chamber.After bonding, the substrate pair may be diced using a diamond saw. Theresulting structure is shown schematically in FIG. 6.

The exact dimensions and structure of the microfluidic chip may bevaried to suit the chip to a particular application. A specific exampleof one possible device according to the present invention is as follows.The device is 4.0 mm square and 0.9 mm thick. The extraction chamber hasa depth of 200 μm and a length and width of 2.8 mm. The fluid ports eachhave a width of 0.4 mm. The device has a dense array of columnsoccupying an area 2.0 mm×2.8 mm within the chamber. The columns have aheight of 200 μm, a cross sectional length of 50 μm, a cross sectionalwidth of 7 μm, a gap distance of 8 μm between adjacent columns in a row,and a center to center spacing of 15 μm. There are roughly 7,000 columnsin the array. Of course, these dimensions are exemplary of just onepossible embodiment and are not intended to limit the scope of theinvention. The specific dimensions of each material of the device may bevaried in alternative embodiments, preferably within the generalguidelines set forth earlier in this description.

The chip may be incorporated into a region of the cartridge with aflexible, polymeric coating, such as a silicone glue. Alternatively, agasket may be fabricated with matching holes to the fluidic ports on thechip and a sealed fluidic assembly made between the microfluidic domain(the chip) and the macrofluidic domain (the cartridge body). The chipmay be pressed tightly and sealed against the gasket material by bondinganother plastic piece over the chip, thus completely encapsulating thechip within the cartridge.

Alternatively, the chip may be fused or welded to the cartridge directlywithout the use of a gasket. In a particularly advantageous embodiment,a portion of the cartridge itself may be the cover for the chip ratherthan using a separate substrate, e.g., the Pyrex™ glass, to form thecover. In this embodiment, the substrate 22 is inserted into thecartridge and sealed to a wall of the cartridge. The wall has holes init forming the fluid ports to the extraction chamber.

One technique used to make integrated chip and plastic cartridges usesrecessed regions in the plastic to accept the silicon/glassmicromachined chip(s). The recessed regions are precisely dimensioned toaccept and accurately locate the silicon/glass chip. This techniqueallows the small silicon/glass microfluidic chip(s) to be easily alignedto the macrofluidic channels, ports, and other fluidic regions moldedinto the plastic. The recess itself may contain a fluid port to connectwith a fluid port on the bottom of the silicon/glass chip.

In addition, the use of recessed regions allows another plastic moldedcomponent to be easily laminated on top of the firstsilicon/glass/plastic assembly. This second technique is especiallysuitable for interfacing the molded fluid paths in the plastic to thesmall microfluidic openings (typically about 0.5 mm in diameter) whichemerge onto the flat surfaces (on either side of the chip) of thesilicon/glass chip. This technique can also provide a convenient meansfor accessing electrical contacts on the microfluidic chip, ifnecessary. In this case, a region in the laminated plastic is left opento allow easy access for wire bonding to the silicon/glass chip.

A third technique is the forming of molded plastic regions that are theinverse shape of anisotropically etched pyramidal pits in (100) silicon.This technique has several advantages. It allows for easy alignmentbetween the silicon and the plastic and at the same time, minimizes thefluid dead volume where the plastic must be connected to ananisotropically etched fluid pit in a silicon chip.

A fourth technique is the use of laminated or patterned adhesive filmsto make fluid-tight seals between the various plastic and silicon/glasspieces. Materials such as polyimide or Mylar® can be formed in very thinsheets (on the order of 0.0254 mm) and coated on both sides withadhesive (curable by ultra violet or by temperature). The adhesive notonly joins the two components, but also forms fluid-tight seals. Suchsheets can be cut or punched into various shapes, thereby providingaccess holes or other shapes, then laminated onto the plastic and/orsilicon/glass. For some applications, screen-printed adhesives may bemore appropriate as fluid-tight seals.

FIG. 15 illustrates one type of integration between a siliconmicrofluidic chip 7 and a recess 3 within a cartridge 1. Theprecisely-dimensioned recess 3 is molded into the middle plastic portion5 into which the chip 7 is inserted. The chip 7 has a glass portion 9and silicon portion 11 and is accessible to wire connection 13. Achannel 15 is molded into the middle plastic portion 5 and lower plasticportion 17. A laminated interface 19 aligns the channel of the middleand lower plastic components. A gasket or an adhesive 93 allows forfluid-tight lamination, sealing, and integration of the plastic portionand silicon-glass chip 7.

FIG. 12 shows an alternative embodiment of the microfabricated chip inwhich the chip has fluid ports 28 and 30 formed in the base substrate 22rather than the top substrate 24. The chip also includes electrodes 48Aand 48B for heating the internal surfaces of the chamber 26. Theelectrodes are preferably positioned on opposite sides of the bottomwall 23 of the extraction chamber 26. The base substrate 22 isfabricated from a thermally conductive material, preferably silicon, sothat the bottom wall 23 and integrally formed columns may be heated byapplying an appropriate voltage across the electrodes 48A and 48B.

As in the previous embodiment, the chip may be used in combination withthe cartridge, as previously described with reference to FIG. 2. Theoperation of the chip is analogous to the operation described above,except that the internal surfaces of the chamber 26 are heated byapplying a voltage across the electrodes 48A and 48B. The bottom wall 23functions as a resistive heating element for heating the chamber 26.

The microfluidic chip of FIG. 12 may be fabricated using a variety oftechniques, including photolithography and/or micromachining. Apreferred method for fabricating the chip will now be described.

A 100 mm, n-type (100), silicon wafer is used as starting material forthe base substrate 22. The wafer preferably has a resistivity of 1 to100 ohm-cm, depending on the desired final resistance between theelectrodes 48A and 48B. The wafer thickness is preferably in the rangeof 350 to 600 μm, depending on the desired structure. Ohmic contacts aremade by using phosphorous ion implantation into regions in the backside,preferably to a depth of 0.2 to 5 μm. Alternatively, a p-type siliconwafer may be used, and the ohmic contacts made using boron ionimplantation. Implantation is followed by heating of the substrate toactivate the dopant.

Next, the fluid ports 28 and 30 are formed by depositing and patterninga suitable masking material, e.g., silicon nitride, onto the backside ofthe wafer and anisotropic etching the silicon using the mask. The waferis then patterned with photoresist on the frontside to obtain an etchmask for the DRIE process. As shown in FIG. 11, the etch mask defines achamber pattern 44 for forming the extraction chamber in the substrate22 and an array of column patterns 46 for forming a corresponding arrayof columns in the substrate. The patterned wafer is then etched using aDRIE process to form the extraction chamber and integral columns. Thewafer is etched to a depth sufficient for the extraction chamber 26 tomeet the fluid ports 28 and 30.

After etching, the remaining photoresist is removed from the wafer, andthe substrate is then oxidized to cover the internal surfaces of thechamber 26 with an oxide layer, preferably 1 to 100 nm thick. Anelectrically conductive material, e.g., aluminum, gold, or copper, isthen deposited and patterned over the doped regions on the backside ofthe substrate to form the electrodes 48A and 48B. The substrate 22 isthen anodically bonded to a cover 24, preferably thin Pyrex™ glass.After bonding, the substrate pair may be diced to form the finalstructure shown in FIG. 12.

FIG. 13 shows a flow-through chip 21 according to another embodiment ofthe invention in which the internal attachment surfaces for capturingand eluting the analyte are formed by one or more solid supportscontained within the chamber 26. As the fluid sample flows through thechamber 26, the analyte contacts and adheres to the solid support. Toelute the analyte, the chamber 26 is heated while an elution fluid isforced to flow through the chamber, thus releasing the analyte from thesolid support into the elution fluid. Suitable solid supports forcapturing the analyte include filters, beads, fibers, membranes, glasswool, filter paper, gels, etc.

In the embodiment of FIG. 13, the solid support comprises glass beads 50packed within the chamber 26. In embodiments that employ beads, fibers,wool, or gels as the solid support, the device preferably includes abarrier 52 disposed in the chamber 26 adjacent the outlet port 30 forpreventing the solid support material from flowing out of the chamber.The barrier 52 may be any suitable retaining membrane or filter, such asa comb filter, for holding the solid support material within the chamber26. Alternatively, the barrier 52 may comprise a plurality of internalstructures, such as columns, formed within the chamber 26 and having asufficiently small spacing to retain the solid support material.

The chip 21 may be used in combination with the cartridges of theinvention to capture and elute target analyte, as previously described.The operation of the chip 21 is analogous to the operation describedabove, except that the analyte capture surfaces in the chamber 26 areprovided by a solid support, such as the beads 50, rather than by anarray of integrally formed microstructures.

The chip 21 may be fabricated using techniques similar to thosedescribed in earlier embodiments, including photolithography andmicromachining. A preferred method for fabricating the chip will now bedescribed. A 100 mm, n-type (100), 0.1 to 0.2 ohm-cm, silicon wafer ispreferably used as starting material for the base substrate 22. Thewafer is patterned with photoresist on the frontside to obtain an etchmask for a DRIE process. The etch mask defines a chamber pattern forforming the chamber 26 in the substrate 22 and a barrier pattern forforming internal barrier structures, preferably closely spaced columns,within the chamber 26. The patterned wafer is then etched using a DRIEprocess to form the chamber 26 and internal barrier structures. Ofcourse, the structures should have a spacing smaller than the diameterof the beads 50 so that they will retain the beads in the chamber 26.

After etching, the remaining photoresist is removed from the wafer, andone or more electrically conductive materials is then deposited andpatterned on the backside of the substrate to form a resistive heatingelement, temperature sensor, and bond pads. The substrate is thenanodically bonded to a glass cover having holes that form the fluidports 28 and 30. The beads 50 may be packed in the chamber 26 before orafter attaching the cover, preferably after the cover is attached. Thebeads 50 are inserted through the inlet port 28. Of course, the barrier52 should be in place before packing the beads 50 to prevent the beadsfrom flowing out of the chamber 26.

FIG. 14 shows a flow-through chip 31 according to another embodiment ofthe invention in which the solid support contained within the chamber 26comprises a membrane or filter 60 for capturing the target analyte. Thechip 31 includes a base substrate 58, a top substrate 54, and a middlesubstrate 56 sandwiched between the top and base substrates. Theextraction chamber 26 is formed in the top and base substrates 54 and58, and the filter 60 is preferably in thermal contact with the heater34. Alternatively, the filter 60 may be disposed in the base substrate58 adjacent the outlet port 30.

The resistive heating element 34 is preferably positioned on the middlesubstrate 56 for heating the chamber 26. The heating element 34 may becovered by a layer 62 of insulating material, e.g., silicon dioxide,silicon carbide, silicon nitride, plastic, glass, glue or otherpolymers, resist, or ceramic, for protecting the heating element 34 fromfluids flowing through the chamber 26. The middle substrate 56 includesholes (not shown in the side view of FIG. 14) disposed around theheating element 34 to permit continuous fluid flow through the chamberfrom the inlet port 28 to the outlet port 30.

The heating element 34 may be a thin film of metal or polysilicon whichis patterned on the substrate 56. Alternatively, the substrate 56 may bea thin plastic flex-circuit having the heating element 34. In anotherembodiment, the heating element 34 may comprise a laminated heatersource, such as an etched foil-heating element, attached to thesubstrate 56. In embodiments where the heater is part of a laminatedstructure, the substrate 56 is the support for the heater. In yetanother embodiment, the substrates 56 and 58, together with the heatingelement 34 and insulator layer 62, may all be fabricated from a singlesubstrate using techniques known to those skilled in the art, e.g., thinfilm processing.

The chip 31 is used in combination with a cartridge of the presentinvention, as previously described. In operation, a fluid sample isforced to flow through the chip. As the fluid sample flows through thechamber 26, target analyte, e.g., nucleic acid, contacts and adheres tothe filter 60. The chamber is optionally washed to remove unwantedparticles. To elute the analyte, the chamber 26 is heated with theheating element 34 while an elution fluid is forced to flow through thechamber, releasing the analyte from the filter 60 into the elutionfluid.

The top and base substrates 54 and 58 are preferably low cost moldedplastic parts, and the middle substrate 56 is preferably a plastic flexcircuit. The device 31 may be fabricated by precutting the filter 60 tosize and then assembling the filter 60 and the substrates 54, 56, and 58using adhesives, such as glue, or by welding, e.g. ultrasonic welding.

FIG. 16 shows another exemplary cartridge of the invention. Thecartridge 161 is comprised of a top portion 163 and bottom portion 165with a middle portion 167 therebetween. The middle portion 167 ispreferably a printed circuit board (or flex circuit) having electricalcircuitry 169. Mating of board 167 with bottom 165 forms one wall of thefluid flow regions. The sample flow path includes, in a downstreamdirection, a lysing chamber 173, a flow-through chip 177, and a ventedwaste chamber 203. The elution flow path includes the flow through chip177, a reagent chamber 179, and a reaction chamber 181.

As shown in FIG. 16 and the detail of FIG. 17, the lysing chamber 173has a chemically treated filter paper 183 which accepts the sample. Acap 185 is connected to the top by a flexible arm 187 and made to coverthe lysing chamber 173 after the sample is added. The cap includes amembrane 189 made of material such as Goretex® which allows thetransmission of gases but prevents the flow of liquid. A desiccant 191is located in the cap on top of the membrane 189. A heater 193 islocated on flex circuit 167 below the sample port and heats the filterpaper 183 and the sample when the cap is in a closed position.

In operation, after the sample is added to the filter paper 183, theheater dries the sample and moisture rises through the membrane 189 andis absorbed into the desiccant 191. At the same time, chemicalsimpregnated in the paper lyse the cells and bind various biologicalmolecules to the paper itself. The cartridge bottom includes a washstorage chamber 195 which is connected by channel 197 to the sample portin an area beneath the filter paper 183. Thus, after the sample isdried, wash fluid is forced to flow from C to D, as depicted in FIG. 17,through the filter paper 183 to wash out and/or elute processingchemicals which are present in the filter paper. The waste processingchemicals and wash are prevented from flowing into the desiccant bymembrane 189 and exit the sample port through outlet D.

As shown in FIG. 16 and the detail of FIG. 18, waste fluid is washedaway from the sample flow path and redirected into waste chamber 201 bya flow diverter 174. The flow diverters 174, 175 may comprise acapillary or hydrophobic membrane to allow fluid to pass when athreshold back pressure develops in the regions before the diverters.The waste fluid filling waste chamber 201 creates pressure in region176. Once the waste chamber 201 is filled with fluid, the pressure inregion 176 triggers the diverter 174 to allow fluid to pass.Simultaneously, the sample in lysing chamber 173 is heated by heater 193causing the nucleic acid to be released from the filter paper 183 andflow out through outlet D.

The sample flows along the sample flow path through diverter 174 andinto chip 177 where target analyte is extracted. Waste componentsflowing from the chip 177 are redirected by flow diverter 175 to flowinto a second waste chamber 203. Waste components collecting in thesecond waste chamber 203 create back pressure in region 178. Once wastecomponents fill the second waste chamber 203, the pressure in region 178is sufficient to release diverter 175 and allow fluid to pass.Simultaneously, a voltage or heat is applied to the chip 177 throughconnectors in the flex circuit 167, releasing the target analyte.Thereby, the analyte flows down the elution flow path and into a reagentchamber 179 where predried reagents are reconstituted and mixed with theanalyte. The mixture continues to flow into and fill the reactionchamber 181. The elution flow path ends at reaction chamber 181 whereamplification, e.g. PCR, takes place.

Historically, the lysis step in sample processing has been a timeconsuming and difficult task, especially for spores and certain cellstructures. In further embodiments, the present invention addresses thisproblem by providing a method and device for the rapid lysing of samplecomponents, e.g., cells, spores, or microorganisms, using ultrasound.The ultrasonic lysing may be performed in a fully integrated cartridge,such as the cartridge of FIG. 2, or may be performed with a cartridgethat performs only lysing of sample components.

FIG. 19 shows an exemplary device for lysing sample components, e.g.,cells, spores, or microorganisms. The device includes a cartridge 70having an inlet port 72 for introducing the sample into the cartridge,and a lysing chamber 74 in fluid communication with the inlet port 72for receiving the sample. The cartridge also includes an outlet port 76for exit of the sample from the chamber 74.

The chamber 74 contains a solid phase for capturing the components ofthe sample to be lysed. Suitable solid phases for capturing cells,spores, or microorganisms include, e.g., filters, beads, fibers,membranes, glass wool, filter paper, polymers and gels. The solid phasemay capture the desired sample components through physical retention,e.g., size exclusion, through affinity retention, or through chemicalselection. In the presently preferred embodiment, the solid phasecomprises a membrane or filter 86 for capturing the components to belysed. Suitable filter materials include glass, fiberglass, nylon, nylonderivatives, cellulose, cellulose derivatives, and other polymers. In analternative embodiment, the solid phase comprises polystyrene, silica,agarose, cellulose, or acrylamide beads.

The device also includes an ultrasonic transducer, such as an ultrasonichorn 88, that is coupled to the cartridge for transferring ultrasonicenergy to the components captured on the solid phase, e.g., captured onfilter 86. A miniature ultrasonic horn is presently preferred as thetransducer because it allows focusing of ultrasonic energy onto thecomponents captured on the solid phase. To this end, it is alsopreferred that the horn 88 be coupled to the cartridge 70 such that thelongitudinal axis of the horn 88 is perpendicular to the filter 86.Additionally, the horn 88 is preferably coupled directly to a wall ofthe chamber 74.

In operation, a sample fluid is introduced into the inlet port 72 andforced to flow into chamber 74. As the sample flows into the chamber 74,the sample components to be lysed are captured by the filter 86. Thesample may be made to flow continually through the chamber 74, or thecartridge 70 may include flow controllers, e.g. valves, for holding thesample fluid in chamber 74 for lysis. Continuous flow processing issuitable for larger sample volumes, e.g. 1 mL or greater, while holdingthe sample in the chamber 74 may be appropriate for smaller samplevolumes, e.g. 100 μl.

The sample components captured on the filter 86 are then lysed bytransferring ultrasonic energy from the horn 88 to the capturedcomponents. The ultrasonic energy causes rapid lysis of cells, spores,or microorganisms captured on the filter. As a specific example, rapidlysis of spores in a 100 μl sample was accomplished by applyingultrasound for thirty seconds at a frequency of 47 kHz and an ultrasonicoutput of 50 watts. Ultrasonic output in the range of 10 to 60 watts ispresently preferred. The ultrasonic lysis may be performed with orwithout the use of lysing reagents, e.g., chaotropes, detergents, salts,and reducing agents. The ultrasonic lysis permits the choice ofbuffer/resuspension solution related to the post lysis protocol (e.g.,buffer that is non-inhibitory to PCR).

Typically, the ultrasonic transducer will be a separate component fromthe cartridge and coupled to the cartridge by an operator or machine.Alternatively, the transducer may be located in an external instrumentthat receives the cartridge for processing. In this embodiment, thetransducer is preferably positioned in the instrument such that itpresses against a wall of the lysing chamber when the cartridge isinserted into the instrument for processing. In another embodiment, thetransducer may be built into the cartridge. In this embodiment, thecartridge includes suitable electrical connectors for connecting thetransducer to a power supply. In embodiments in which the transducer isbuilt into the cartridge, the transducer should be prevented fromcontacting the fluid sample directly, e.g., the transducer should belaminated or separated from the sample by a chamber wall.

The cartridge 70 may be fabricated using techniques previously describedfor the cartridge of FIG. 2. In particular, the cartridge 70 preferablycomprises first and second molded plastic parts 78 and 80 which supportfilter 86. Filter 86 may optionally be heat sealed to the plastic parts78 and 80. The cartridge also includes first and second plastic films 82and 84 sealed to parts 78 and 80, respectively. Examples of suitablematerials for the plastic parts 78 and 80 and for the films 82 and 84include, e.g., polycarbonate, polystyrene, polypropylene, polyethylene,acrylic, and commercial polymers. To aid in the transfer of ultrasonicenergy to the sample components, it is preferred that films 82 and 84 berelatively thin. Films 82 and 84 preferably have a thickness in therange of 0.01 to 0.5 mm, and more preferably have a thickness of about0.05 mm.

FIG. 20 shows another embodiment of a cartridge for ultrasonicallylysing sample components. The cartridge 90 includes beads 94 in itslysing chamber for rupturing the components captured on the solid phase.The cartridge 90 also includes an ultrasonic transducer 92 in the formof a disk coupled to a wall of the chamber. In operation, the transducer92 transfers ultrasonic energy to the captured sample components toeffect lysing. The ultrasonic energy also agitates the beads so that thebeads rupture the sample components to effect lysing. Suitable beads forrupturing sample components include polystyrene and silica. The beadsmay be porous or non-porous and preferably have a diameter in the rangeof 1 to 200 μm. As a specific example, the ultrasonic lysis chamber mayhave a volume capacity of 110 μL and contain 10 μL of glass beads.

Although the embodiments of FIGS. 19 and 20 show cartridges that performonly lysing functions, it is to be understood that the ultrasonic lysisof the present invention may be incorporated into cartridges thatperform a variety of other function. For example, referring again toFIG. 2, an ultrasonic transducer may be coupled to the lysing chamber119 to lyse cells, spores, or microorganisms in a fluid sample. Further,beads could also be put in the chamber 119 to rupture the samplecomponents. In another embodiment, a heating element may be used inplace of or in combination with an ultrasonic transducer to lyse samplecomponents captured on a solid phase.

Although the above description contains many specificities, these shouldnot be construed as limitations on the scope of the invention, butmerely as illustrations of some of the presently preferred embodiments.Many possible variations and modifications to the invention will beapparent to one skilled in the art upon consideration of thisdisclosure. Therefore, the scope of the invention should be determinedby the following claims and their legal equivalents.

1. A method for extracting nucleic acid from a sample, the samplecontaining cells, spores, or microorganisms, the method comprising: a)introducing the sample into a cartridge having: i) a lysing region forlysing the cells, spores, or microorganisms to release the nucleic acidtherefrom; and ii) a capture region, the capture region comprising achannel or chamber containing capture material for capturing the nucleicacid; b) drying the sample on paper or membrane material in the lysingregion, the paper or membrane material being impregnated with at leastone chemical for lysing the cells, spores, or microorganisms in thesample to release the nucleic acid from the cells, spores, ormicroorganisms, while binding contaminants or inhibitors in the sampleto the paper or membrane material; c) placing fluid in the lysing regionand releasing the nucleic acid from the paper or membrane material intothe fluid while the contaminants or inhibitors remain bound to the paperor membrane material; d) forcing the fluid containing the nucleic acidto flow through the capture region to capture the nucleic acid with thecapture material; and e) eluting the nucleic acid from the captureregion.
 2. The method of claim 1, wherein the cartridge further includesa reaction chamber, and the method further comprises the steps of: i)forcing the nucleic acid eluted from the capture region to flow into thereaction chamber; ii) amplifying the nucleic acid in the reactionchamber; and iii) detecting the amplified nucleic acid.
 3. The method ofclaim 2, wherein the amplification requires temperature control of thereaction chamber, the portion of the cartridge defining the reactionchamber protrudes from the rest of the cartridge body, and thetemperature of the reaction chamber is controlled by inserting thereaction chamber into a thermal sleeve and heating or cooling thereaction chamber according to a time/temperature profile.
 4. The methodof claim 3, wherein the cartridge further includes a reagent chambercontaining dried or lyophilized reagents, and the method furthercomprises the step of mixing the nucleic acid with the reagents in thereagent chamber prior to forcing the nucleic acid to flow into thereaction chamber.
 5. The method of claim 1, further comprising the stepsof: i) forcing the nucleic acid eluted from the capture region to flowinto a reaction vessel coupled to the cartridge; ii) amplifying thenucleic acid in the reaction vessel; and iii) detecting the amplifiednucleic acid.
 6. The method of claim 5, wherein the amplificationrequires temperature control of the reaction vessel, and wherein thetemperature of the vessel is controlled by inserting the vessel into athermal sleeve and heating or cooling the vessel according to atime/temperature profile.
 7. The method of claim 5, wherein thecartridge further includes a reagent chamber containing dried orlyophilized reagents, and the method further comprises the step ofmixing the eluted nucleic acid with the reagents in the reagent chamberprior to forcing the nucleic acid to flow into the reaction vessel. 8.The method of claim 1, wherein the capture material comprises at leastone solid support selected from the group consisting of filters,membranes, beads, fiber, glass wool, filter paper, polymers, and gel. 9.The method of claim 1, wherein the capture region comprises anextraction chamber formed in a microfluidic chip, and wherein thecapture material comprises an array of microstructures extending intothe extraction chamber, each of the microstructures having an aspectratio (height to width) of at least 2:1.
 10. The method of claim 1,wherein the cartridge includes at least one waste chamber for receivingwaste fluid and at least one additional chamber for receiving thenucleic acid eluted from the capture region, the fluid forced to flowthrough the capture region in d) is forced to flow into the at least onewaste chamber after flowing through the capture region, and the nucleicacid is eluted from the capture region by forcing elution fluid to flowthrough the capture region and into the additional chamber.
 11. Themethod of claim 10, wherein the volume of sample placed in the lysingregion is greater than the volume of elution fluid forced to flowthrough the capture region, whereby the nucleic acid extracted from thesample is concentrated in the smaller volume of elution fluid.
 12. Themethod of claim 10, wherein the ratio of the volume of fluid forced toflow through the capture region in d) to the volume capacity of thecapture region is at least 2:1.
 13. The method of claim 10, wherein theadditional chamber comprises a reagent chamber containing reagents, andthe method further comprises the mixing the nucleic acid with thereagents in the reagent chamber.
 14. The method of claim 10, wherein theadditional chamber comprises a reaction chamber, and the method furthercomprises the step of amplifying the nucleic acid in the reactionchamber.
 15. The method of claim 1, wherein the cartridge includes atleast one waste chamber for receiving waste fluid, the fluid forced toflow through the capture region in d) is forced to flow into the atleast one waste chamber after flowing through the capture region, andthe nucleic acid is eluted from the capture region by forcing elutionfluid to flow through the capture region and into a reaction vesselcoupled to the cartridge.
 16. The method of claim 15, wherein the volumeof sample placed in the lysing region is greater than the volume ofelution fluid forced to flow through the capture region, whereby thenucleic acid extracted from the sample is concentrated in the smallervolume of elution fluid.
 17. The method of claim 15, wherein the ratioof the volume of fluid forced to flow through the capture region in d)to the volume capacity of the capture region is at least 2:1.
 18. Themethod of claim 15, wherein the cartridge includes a reagent chamber,and the method further comprises the step of mixing the nucleic acideluted from the capture region with reagents in the reagent chamberprior to forcing the nucleic acid to flow into the reaction vessel. 19.The method of claim 15, further comprising the step of amplifying anddetecting the nucleic acid in the reaction vessel.
 20. The method ofclaim 1, wherein the cartridge includes a desiccant adjacent the lysingregion, and wherein the sample is dried on the paper or membranematerial by heating the sample and absorbing moisture with thedesiccant.
 21. The method of claim 1, wherein the nucleic acid isreleased from the paper or membrane material into the fluid in thelysing region by heating the paper or membrane material.
 22. The methodof claim 1, wherein the cartridge includes at least one waste chamber,and wherein c) step (c) is preceded by the additional steps of bindingthe nucleic acid released from the cells, spores, or microorganisms tothe paper or membrane material, washing the lysing region with washfluid, and forcing the wash fluid to flow out of the lysing region andinto the at least one waste chamber while the nucleic acid remains boundto the paper or membrane material.
 23. The method of claim 1, whereinthe chemical comprises at least one lysing agent selected from the groupconsisting of enzymes, detergents, and chaotropes.
 24. The method ofclaim 1, wherein the chemical comprises a chaotropic salt.
 25. Themethod of claim 1, wherein the paper or membrane material comprisescellulose, nitrocellulose, polycarbonate, or nylon.